Physical and surface properties of insect apolipophorin III

Apolipophorin III (apoLp-III) from Manduca sexta has a molecular weight of 18,100. Based on its hydrodynamic properties (sedimentation and diffusion coefficients, frictional ratio, intrinsic viscosity) and its behavior during gel permeation chromatography, we concluded that apoLp-III is a prolate ellipsoid with an axial ratio of about 3. The circular dichroic spectrum of apoLp-III suggests that the protein contains approximately 50% alpha-helix. At the air-water interface, apoLp-III forms a monolayer which is gaseous at surface pressures less than or equal to 1 dyne/cm. The isotherm of this phase yields an excluded molecular area of 3800 A2/molecule (23 A2/amino acid). At a surface pressure of 22.1 dynes/cm, the monolayer undergoes a phase transition reminiscent of a first-order phase transition of pure lipids. The monolayer can be compressed in this surface pressure range to an area per molecule of 480 A2 (2.9 A2/amino acid). Since a globular protein of molecular weight 18,100 could occupy an area of only about 2000 A2 when bound to a surface, it is suggested that in the expanded state, apoLp-III must unfold on the surface, whereas in the compressed state, the molecule is oriented with its minor axis parallel to the water surface. ApoLp-III binds with high affinity (Kd = 1.9 X 10(-7)M) to both phosphatidylcholine- and diacylglycerol-coated polystyrene beads. All of these results are consistent with the proposal that apoLp-III plays a key role in increasing the capacity of the insect lipoprotein, lipophorin, to transport diacylglycerol by stabilizing the increment of lipid-water interface that results from diacylglycerol uptake.     

Epiphyseal-based designs for tibial plateau components--II. Stress analysis in the sagittal plane

A two-dimensional, finite element study was undertaken to establish the stresses in the proximal tibia before and after total knee arthroplasty. Equivalent-thickness models in a sagittal plane were created for the natural, proximal tibia and for the proximal tibia with two different types of tibial plateau components. All components simulated bony ingrowth fixation, i.e. no cement layer existed between component and bone. In addition, the interface between component and bone was assumed to be intimately connected, representing complete bony ingrowth and a rigid state of fixation. Two load cases were considered: a joint reaction force acting in conjunction with a patellar ligament force, simulating the knee at 40 degrees of flexion; and a joint reaction force directed along the long axis of the tibia. For the natural tibia model, the pattern of principal stresses for loadcase 1 more closely corresponds to the epiphyseal plate geometry and trabecular morphology than do the principal stress patterns for loadcase 2. Judging from the distribution of principal stresses, loadcase 1 represents a more severe test of implant design than does loadcase 2. The model of the component with a peg predicted that the trabecular bone near the tip of the peg will experience higher than normal stresses, while the bone stresses near the posterior aspect adjacent to the metal tray will be reduced. A component without pegs that incorporates a posterior chamfer and an anterior lip lead to stress distributions closer to those existing in the natural tibia. The interface geometry for this design is based upon the contour of the epiphyseal plate.     

Alpha-thrombin-induced inositol phosphate formation in G0-arrested and cycling hamster lung fibroblasts: evidence for a protein kinase C-mediated desensitization response

In resting Chinese hamster fibroblasts (CCL39) alpha-thrombin rapidly induces the breakdown of phosphoinositides. Accumulation of inositol phosphates (IP), measured in the presence of Li+, is detectable within 5s (seconds) of thrombin stimulation. Formation of inositol tris- and bisphosphates slightly precedes that of inositol monophosphate, indicating that thrombin activates primarily the phospholipase C-mediated generation of inositol trisphosphate from phosphatidylinositol 4,5-bisphosphate. Initial rates of IP production increase with thrombin concentration, with no apparent saturability over the range 10(-4)-10 U/ml. Thrombin-induced phosphoinositide hydrolysis rapidly desensitizes (t1/2 less than 5 min), but a residual activity, corresponding to about 10% of the initial stimulation is sustained for at least 9 h, in contrast with the undetectable activity of G0-arrested cells. This apparent desensitization may be due to a feedback regulation by protein kinase C, since pretreatment with the phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA) markedly inhibits (by up to 70%) subsequent thrombin-induced inositol phosphate formation. Conversely, growth factor deprivation of CCL39 cells results in a progressive increase of thrombin-induced phosphoinositide hydrolysis, from the very low level of exponentially growing cells to the maximal level of G0-arrested cells. This "up regulation" was found maximal in A51, a very well growth-arrested CCL39 derivative, and reduced or virtually abolished in two tumoral and growth factor-relaxed derivatives of CCL39. Although preliminary, this observation suggests that a persistent activation of phosphatidyl inositol breakdown might operate in variants selected for autonomous growth.     

Respiratory-related cortical potentials evoked by inspiratory occlusion in humans

It has long been recognized that humans can perceive respiratory loads. There have been several studies on the detection and psychophysical quantification of mechanical load perception. This investigation was designed to record cortical sensory neurogenic activity related to inspiratory mechanical loading in humans. Inspiration was periodically occluded in human subjects while the electroencephalographic (EEG) activity in the somatosensory region of the cerebral cortex was recorded. The onset of inspiratory mouth pressure (Pm) was used to initiate signal averaging of the EEG signals. Cortical evoked potentials elicited by inspiratory occlusions were observed when C3 and C alpha were referenced to CZ. This evoked potential was not observed with the control (unoccluded) breaths. There was considerable subject variability in the peak latencies that was related to the differences in the inspiratory drive, as measured by occlusion pressure (P0.1). The results of this study demonstrate that neurogenic activity can be recorded in the somatosensory region of the cortex that is related to inspiratory occlusions. The peak latencies are longer than analogous somatosensory evoked potentials elicited by stimulation of the hand and foot. It is hypothesized that a portion of this latency difference is related to the time required for the subject to generate sufficient inspiratory force to activate the afferents mediating the cortical response.     

Role of protein synthesis and proteases in production and inactivation of maturation-promoting activity during meiotic maturation of starfish oocytes

In starfish oocytes, activity of the maturation-promoting factor (MPF) and that of a major cAMP-independent protein kinase dropped at the time of meiotic cleavage, and rose again after the first but not the second meiotic cleavage. Protein synthesis was required before the first meiotic cleavage for both MPF and protein kinase activity to rise again after the first meiotic cleavage. Microinjection of either leupeptin or soybean trypsin inhibitor early enough prior to first polar body emission suppressed both the meiotic cleavage and the associated drop of MPF activity. Microinjection of leupeptin or soybean trypsin inhibitor during the 10-min period before the first meiotic cleavage also suppressed cytokinesis but did not prevent a decrease in MPF activity at the normal time of cytokinesis. The lysosomotropic inhibitor ammonia neither suppressed cytokinesis nor the drop of MPF activity at the time of first meiotic cleavage. Activity of neutral proteases sensitive to leupeptin and soybean trypsin inhibitor was demonstrated in oocyte homogenates prepared at the time of first meiotic cleavage. It is proposed that such proteases might be involved in degradation of protein kinase(s) and in the drop of MPF activity at the time of first meiotic cleavage.     

Reorganization of intermediate filament cytoskeleton in induced metanephric mesenchyme cells is independent of tubule morphogenesis

The metanephric mesenchyme becomes converted into epithelial tubules if cultured in transfilter contact with an inductor tissue. The expression of intermediate filaments (IFs), used as cell-type-specific markers has been studied in this model system for differentiation and organogenesis. In immunofluorescence microscopy of frozen sections, the undifferentiated cells of isolated metanephric mesenchymes uniformly showed IFs of vimentin type only. Also, when cultured as a monolayer, cells from the uninduced mesenchymes showed only vimentin filaments. In frozen sections of transfilter explants, epithelial tubules apparently negative for vimentin could be seen after 3 days in culture, but expression of cytokeratin could not be demonstrated in the developing tubules until the fourth day of culture. Sections of explants cultured further showed tubule cells with distinct fibrillar cytokeratin positivity. The appearance of cytokeratin in the explants was also demonstrated with immunoblotting experiments, using two different cytokeratin antibodies. Expression of IFs was further examined in monolayer cultures of metanephric mesenchymes which had been initially exposed to a short transfilter induction pulse. In these experiments, cytokeratin-positive cells could be demonstrated after a total of 4 days in culture. Double immunofluorescence experiments showed varying amounts of vimentin in the cytokeratin-positive cells: after 4 days in culture, most cytokeratin-positive cells still showed vimentin-positivity although often in a nonfibrillar form. During further culture, gradual disappearance of vimentin-specific fluorescence was observed in cytokeratin-positive cells. The results suggest that the vimentin-positive metanephric mesenchyme cells lose their fibrillar vimentin organization upon induction that leads to kidney tubule formation. This change may be essential for the transformation from an undifferentiated mesenchymal cell into a specialized epithelial cell. Cytokeratin filaments, regarded as a marker for epithelial cells, seem to appear simultaneously with or soon after the change in vimentin organization. These changes in IF expression also occur in monolayer cultures of mesenchyme cells initially exposed to a short transfilter induction pulse. This suggests that epithelial differentiation, as revealed by the emergence of cytokeratin positivity, may occur even in the absence of a clear morphological differentiation and three-dimensional organization of the cells.     

Phospholipid methyltransferase phosphorylation by intact hepatocytes: effect of glucagon

We have obtained a rabbit antiserum that specifically immunoprecipitates the 50K and 25K proteins of rat liver phospholipid methyltransferase. Exposure of intact rat hepatocytes preincubated with [32P]phosphate to glucagon induces a time-dependent phosphorylation of the 50K protein of phospholipid methyltransferase. The incorporation of 32P into the 50K protein was only on phosphoserine. These data support the concept that the activation of rat liver phospholipid methyltransferase by glucagon is mediated by phosphorylation of the enzyme.     

Sequential homologies between procarboxypeptidases A and B from porcine pancreas

Automated Edman degradation of monomeric procarboxypeptidases A and B from porcine pancreas shows that their N-terminal regions (from residue 1 to 34-37) present a high degree of sequential homology to each other as well as to other related procarboxypeptidases. Conformational predictions based on these sequences confirm their structural homology and indicate the probable existence of two beta-turns, one beta-chain and a long alpha-helix in them. On the other hand, tryptic peptide maps on a reverse-phase column indicate great sequential similarities (if not identity) between monomeric procarboxypeptidase A and the procarboxypeptidase A subunit isolated from its binary complex with proproteinase E.