Redistribution of pulmonary blood flow during unilateral hypoxia in prone and supine dogs

We usedfluorescent-labeled microspheres in pentobarbital-anesthetized dogs tostudy the effects of unilateral alveolar hypoxia on the pulmonary bloodflow distribution. The left lung was ventilated with inspiredO₂ fraction of 1.0, 0.09, or 0.03 in random order; the right lung was ventilated with inspiredO₂ fraction of 1.0. The lungs wereremoved, cleared of blood, dried at total lung capacity, then cubed toobtain ~1,500 small pieces of lung (~1.7 cm³). The coefficient ofvariation of flow increased (P < 0.001) in the hypoxic lung but was unchanged in the hyperoxic lung.Most (70-80%) variance in flow in the hyperoxic lung wasattributable to structure, in contrast to only 30-40% of thevariance in flow in the hypoxic lung(P < 0.001). When adjusted for thechange in total flow to each lung, 90-95% of the variance in thehyperoxic lung was attributable to structure compared with 70-80%in the hypoxic lung (P < 0.001). Thehilar-to-peripheral gradient, adjusted for change in total flow,decreased in the hypoxic lung (P = 0.005) but did not change in the hyperoxic lung. We conclude thathypoxic vasoconstriction alters the regional distribution of flow inthe hypoxic, but not in the hyperoxic, lung.

     

Properties of two multifunctional plant fatty acid acetylenase/desaturase enzymes.

The properties of the Delta6 desaturase/acetylenase from the moss Ceratodon purpureus and the Delta12 acetylenase from the dicot Crepis alpina were studied by expressing the encoding genes in Arabidopsis thaliana and Saccharomyces cerevisiae. The acetylenase from C. alpinaDelta12 desaturated both oleate and linoleate with about equal efficiency. The desaturation of oleate gave rise to 9(Z),12(E)- and 9(Z),12(Z)-octadecadienoates in a ratio of approximately 3 : 1. Experiments using stereospecifically deuterated oleates showed that the pro-R hydrogen atoms were removed from C-12 and C-13 in the introduction of the 12(Z) double bond, whereas the pro-R and pro-S hydrogen atoms were removed from these carbons during the formation of the 12(E) double bond. The results suggested that the Delta12 acetylenase could accommodate oleate having either a cisoid or transoid conformation of the C(12)-C(13) single bond, and that these conformers served as precursors of the 12(Z) and 12(E) double bonds, respectively. However, only the 9(Z),12(Z)-octadecadienoate isomer could be further desaturated to 9(Z)-octadecen-12-ynoate (crepenynate) by the enzyme. The evolutionarily closely related Delta12 epoxygenase from Crepis palaestina had only weak desaturase activity but could also produce 9(Z),12(E)-octadecadienoate from oleate. The Delta6 acetylenase/desaturase from C. purpureus, on the other hand, produced only the 6(Z) isomers using C16 and C18 acyl groups possessing a Delta9 double bond as substrates. The Delta6 double bond was efficiently further converted to an acetylenic bond by a second round of desaturation but only if the acyl substrate had a Delta12 double bond and that this was in the Z configuration.     

Pulmonary blood flow distribution has a hilar-to-peripheral gradient in awake, prone sheep

Walther, Sten M., Karen B. Domino, Robb W. Glenny, Nayak L. Polissar, and Michael P. Hlastala. Pulmonary blood flow distribution has a hilar-to-peripheral gradient in awake, prone sheep.J. Appl. Physiol. 82(2): 678-685, 1997.We examined the pulmonary blood flow distribution withintravenous fluorescent microspheres (15 µm) in nine prone,unanesthetized, lambs. Lungs flushed free of blood were air-dried attotal lung capacity and sectioned into~2-cm³ pieces. The pieces wereweighed, identified by lobe, and assigned spatial coordinates.Fluorescence was read on a spectrophotometer, and signals werecorrected for piece weight and normalized to mean flow. Pulmonary bloodflow heterogeneity was assessed by using the coefficient of variationof the flow data. The number of pieces (±SD) analyzed were 1,249 ± 150/animal. Heterogeneity of blood flow was 29.5 ± 6.5%(coefficient of variation = SD/mean). Pulmonary blood flow decreasedwith distance from hilus (P < 0.002) but did not change significantly with vertical height. Distance fromthe hilus was the best predictor of pulmonary blood flow (R² = 0.201) and,together with spatial coordinates and lobe, accounted for 33.7 ± 12.0% of blood flow variability. We conclude that pulmonary blood flowin the awake, prone sheep is distributed with a hilar-to-peripheral gradient but no significant vertical gradient.

     

A family of eukaryotic lysophospholipid acyltransferases with broad specificity

The budding yeast ALE1 gene encodes a lysophospholipid acyltransferase (LPLAT) with broad specificity. We show that yeast LPLAT (ScLPLAT) belongs to a distinct protein family that includes human MBOAT1, MBOAT2, MBOAT4, and several closely related proteins from other eukaryotes. We further show that two plant proteins within this family, the Arabidopsis proteins AtLPLAT1 and AtLPLAT2, possess lysophospholipid acyltransferase activities similar to ScLPLAT. We propose that other members of this protein family, which we refer to as the LPLAT family, also are likely to possess LPLAT activity. Finally, we show that ScLPLAT differs from the specific lysophosphatidic acid acyltransferase that is encoded by SLC1 in that it cannot efficiently use lysophosphatidic acid produced by acylation of glycerol-3-phosphate in vitro.     

Modeling of Substance P and 5-HT Induced Synaptic Plasticity in the Lamprey Spinal CPG: Consequences for Network Pattern Generation

Consequences of synaptic plasticity in the lamprey spinal CPG are analyzed by means of simulations. This is motivated by the effects substance P (a tachykinin) and serotonin (5-hydroxytryptamin; 5-HT) have on synaptic transmission in the locomotor network. Activity-dependent synaptic depression and potentiation have recently been shown experimentally using paired intracellular recordings. Although normally activity-dependent plasticity presumably does not contribute to the patterning of network activity, this changes in the presence of the neuromodulators substance P and 5-HT, which evoke significant plasticity. Substance P can induce a faster and larger depression of inhibitory connections but potentiation of excitatory inputs, whereas 5-HT induces facilitation of both inhibitory and excitatory inputs. Changes in the amplitude of the first postsynaptic potential are also seen. These changes could thus be a potential mechanism underlying the modulatory role these substances have on the rhythmic network activity.The aim of the present study has been to implement the activity dependent synaptic depression and facilitation induced by substance P and 5-HT into two alternative models of the lamprey spinal locomotor network, one relying on reciprocal inhibition for bursting and one in which each hemicord is capable of oscillations. The consequences of the plasticity of inhibitory and excitatory connections are then explored on the network level.In the intact spinal cord, tachykinins and 5-HT, which can be endogenously released, increase and decrease the frequency of the alternating left-right burst pattern, respectively. The frequency decreasing effect of 5-HT has previously been explained based on its conductance decreasing effect on K _Ca underlying the postspike afterhyperpolarization (AHP). The present simulations show that short-term synaptic plasticity may have strong effects on frequency regulation in the lamprey spinal CPG. In the network model relying on reciprocal inhibition, the observed effects substance P and 5-HT have on network behavior (i.e., a frequency increase and decrease respectively) can to a substantial part be explained by their effects on the total extent and time dynamics of synaptic depression and facilitation. The cellular effects of these substances will in the 5-HT case further contribute to its network effect.     

Soil conditions and regeneration after clear felling of a Pinus sylvestris L. stand in a nitrogen experiment,Central Sweden

Forest N fertilization is a common practice in areas of Sweden that are not affected by high levels of N deposition. The environmental consequences of high N input to closed forests are fairly well known, but the long-term effects following clear-felling are a lot less well known. Thus, residual effects on soil and planted seedlings of previous N additions at an experimental N gradient 11 years after clear-felling were studied at a naturally nutrient-poor forest site in central Sweden. The experimental N gradient had been established by three repeated applications (in 1967, 1974 and 1981) of six dosages of NH₄NO₃ with increments of 120 kg N ha^–1. Thus, in total, the applied N dose ranged between 0 and 1800 kg N ha^–1. The study examined extractable base cations and P, soil pH, total-N, total-C, net N-mineralization and potential nitrification in four soil horizons (the humus layer, and 0–5, 5–10 and 10–20 cm in the mineral soil). We also measured the survival and growth of planted Pinus sylvestris L. seedlings. The applied N had no effect on the amounts of extractable-P or base cations in the soil. The soil pH decreased with increasing N dose in the deeper soil horizons, while in the humus the pH showed a weak but statistically significant increase due to the N application. Both total-C and total-N increased as a result of the N application, while the C/N ratio decreased. In the humus layer and the uppermost mineral soil layer NH₄ ⁺ was the major inorganic N source, in contrast to the lowest mineral soil horizon where NO₃ ^– dominated. For most of the studied horizons, there was a positive linear relationship between applied N dose and amount of inorganic N. Both net N-mineralization and potential nitrification showed increases with increasing N dose. As for the plants, no difference in survival or growth was found between the different N treatments. For doses generally applied in forest fertilization no significant differences in any of the studied properties were found.     

Detection of weakly interacting anti-carbohydrate scFv phages using surface plasmon resonance

Methods to characterise and confirm specificity of scFv displayed on phages are important during panning procedures, especially when selecting for antibody fragments with weak affinities in the millimole to micromole range. In this report the surface plasmon resonance (SPR) biosensor was used to study and verify specificity of phages displaying weak anti-carbohydrate scFvs. The variable immunoglobulin light (VL) and heavy (VH) chain genes of the weak monoclonal antibody 39.5 were amplified and cloned into a phagemid and displayed as a scFv-pIII fusion protein on filamentous phage. This monoclonal antibody recognises with weak affinity the structural sequence Glcalpha1-4Glc present in a variety of carbohydrate molecules. Injection of the 39.5 phages over a biosensor chip immobilised with a (Glc)4-BSA conjugate confirmed selective binding of the scFv to its antigen. Inhibition studies verified the specificity. These results clearly show that SPR technology can be used to evaluate in terms of binding and specificity weakly interacting scFv displayed on the phage surface.     

Deficiency of oncoretrovirally transduced hematopoietic stem cells and correction through ex vivo expansion

BACKGROUND: Extensive efforts to develop hematopoietic stem cell (HSC) based gene therapy have been hampered by low gene marking. Major emphasis has so far been directed at improving gene transfer efficiency, but low gene marking in transplanted recipients might equally well reflect compromised repopulating activity of transduced cells, competing for reconstitution with endogenous and unmanipulated stem cells. METHODS: The autologous settings of clinical gene therapy protocols preclude evaluation of changes in repopulating ability following transduction; however, using a congenic mouse model, allowing for direct evaluation of gene marking of lympho-myeloid progeny, we show here that these issues can be accurately addressed. RESULTS: We demonstrate that conditions supporting in vitro stem cell self-renewal efficiently promote oncoretroviral-mediated gene transfer to multipotent adult bone marrow stem cells, without prior in vivo conditioning. Despite using optimized culture conditions, transduction resulted in striking losses of repopulating activity, translating into low numbers of gene marked cells in competitively repopulated mice. Subjecting transduced HSCs to an ex vivo expansion protocol following the transduction procedure could partially reverse this loss. CONCLUSIONS: These studies suggest that loss of repopulating ability of transduced HSCs rather than low gene transfer efficiency might be the main problem in clinical gene therapy protocols, and that a clinically feasible ex vivo expansion approach post-transduction can markedly improve reconstitution with gene marked stem cells.     

Integrative neuroscience: linking levels of analyses

If we are to understand how the brain performs different integrated functions in cellular terms, we need both to understand all relevant levels of analysis from the molecular to the behavioural and cognitive levels and to realize an integration of such levels. This is currently a major challenge for neuroscience. Most research, whether dealing with perception, action or learning, focuses on a few levels of organization, for instance the molecular level and brain imaging, and leaves other crucial areas practically untouched. To reach the level of understanding that we desire, a multi-level approach is required in which the different levels link into each other. It is possible to bridge across the different levels for one system, and this has been demonstrated, for example, in the lamprey in generation of goal-directed locomotion. It can be argued that an integrated analysis of any neural system cannot be performed without the aid of a close interaction between experiments and modelling. The dynamic processing within any neural system is such that an intuitive interpretation is rarely sufficient.