Identifying individual scaffolding molecules in the postsynaptic density.

Extended abstract of a paper presented at Microscopy and Microanalysis 2008 in Albuquerque, New Mexico, USA, August 3 - August 7, 2008.

Visual assessment of hemolysis in red blood cell units and segments can be deceptive.

BACKGROUND: Blood components that appear hemolyzed are discarded. However, visual inspection is subjective and criteria for excessive hemolysis are poorly defined. STUDY DESIGN AND METHODS: Packed RBCs (10 CPDA-1, 10 Adsol) were collected. Half of each unit was leukoreduced. Plasma Hb was measured and compared in segments and units by three methods: 1) a HemoCue Plasma/Low Hb Photometer system; 2) a tetramethyl-benzidine (TMB) chemical method, and 3) a free Hb visual comparator. RESULTS: Visual assessment tended to overestimate hemolysis. Chemical methods were comparable (r(2)= 0.894; HemoCue = 0.043 +[0.770]x TMB; n = 400; range, 0.01-0.5 g/dL), although the mean plasma Hb (g/dL) for the HemoCue method was higher than that of the TMB method (0.12 vs. 0.10 g/dL, respectively; p < 0.001). No units would have been discarded based on a hemolysis level of at least 0.6 g/dL (approx. 1%) if measured by a chemical method. However, 50 percent of CPDA-1 and 10 percent of Adsol units would have been discarded if only visual criteria were used. Leukoreduction did not increase plasma Hb levels. Discrepancies in plasma Hb levels were noted between units and their corresponding segments. CONCLUSION: Visual assessment of hemolysis can result in unnecessary wastage of blood components. HemoCue offers an alternative, objective method to assess plasma Hb in the setting of blood collection and processing facilities for routine quality control and process validation, and may aid in the development of objective criteria for excessive hemolysis in blood components.

Platelet function abnormalities in qualified whole-blood donors: effects of medication and recent food intake.

BACKGROUND AND OBJECTIVES: Platelet function abnormalities have been reported in blood donors who have not consumed aspirin. Our objective was to identify factors other than aspirin that may contribute to impaired platelet function in qualified volunteer blood donors. MATERIALS AND METHODS: Blood samples were obtained from 24 donors following routine blood donation. Donors completed a study questionnaire that included questions about recent food consumption, medication and medical history. Platelet activation was measured using monoclonal antibodies and flow cytometry. CD62P expression and PAC-1 binding on platelets were used as indicators of platelet activation. Platelet function was measured on a platelet function analyser (PFA-100) using both collagen/epinephrine (cEPI) and collagen/ADP (cADP) cartridges. RESULTS: Fifty-four per cent of donors (13 of 24) had normal platelet function. Thirty-eight per cent (nine of 24) had prolonged cEPI closure times, of whom four (17%) had no cEPI closure (> 300 seconds). No closure was associated with aspirin use (two donors) or chocolate consumption (two donors) before donation. Two donors (8%) had either a shortened cEPI or cADP closure time. CONCLUSIONS: Platelet dysfunction in qualified blood donors is underestimated. Platelet function screening can identify donors with diet-related platelet dysfunction or with poor recollection of aspirin use.

Synapse-forming axons and recombinant agrin induce microprocess formation on myotubes.

We examined cell-surface behavior at nerve-muscle contacts during synaptogenesis in cocultures of rat ventral spinal cord (VSC) neurons and myotubes. Developing synapses in 1-d-old cocultures were identified by the presence of axon-induced acetylcholine receptor (AChR) aggregation. Identified regions were then examined by transmission and scanning electron microscopy. The myotube surface near contacts with axons that induced AChR aggregation typically displayed ruffles, microvilli, and filopodia (microprocesses), indicating motility of the myotube surface. At some of these contact sites microprocesses were wrapped around the axon, resulting in the partial or total "submersion" of the axon within the myotube contours. Sites of myotube contact with somata and dendrites of the same neurons showed much less evidence of motility and surface interaction than sites of contact with axons. Moreover, the distance between opposed membranes of axons and myotubes was smaller than between dendrites or somata and myotubes, suggesting stronger adhesion of axons. These results suggest polarized expression of molecules involved in the induction of microprocess formation and adhesion in developing VSC neurons. We therefore tested the ability of agrin, which is preferentially secreted by axons, to induce microprocess formation in myotubes. Addition of recombinant C-terminal agrin to culture medium resulted in formation of microprocesses within 3 hr. Myotubes transfected with full-length rat agrin constructs displayed numerous filopodia, as revealed by fluorescence microscopy. The results suggest that the induction of muscle cell surface motility may be linked to the signaling processes that trigger the initial formation of the neuromuscular junction.

Preload dependence of fiber shortening rate in conscious dogs with left ventricular hypertrophy.

Employing the new concept of systolic myocardial stiffness, this study addresses the questions of linearity of the end-systolic stress-strain relations in left ventricular hypertrophy and the preload dependence of fiber shortening rate. Pressure overload hypertrophy was induced in six puppies by banding the ascending aorta. Ultrasonic crystals were implanted for measurement of short axis and wall thickness in the six dogs with hypertrophy and in five control dogs. A pressure catheter was inserted through the apex for left ventricular pressure measurement. Load was altered by graded infusions of phenylephrine in the setting of beta-adrenergic blockade. Linearity of the end-systolic stress-strain relations was observed in all cases, and preload-corrected shortening rate-afterload relations were derived from these stress-strain relations. Without preload correction, mid wall and endocardial shortening rate were depressed (p less than 0.05 and p less than 0.005, respectively) in the hypertrophy group. However, with preload correction at 35 g/cm2, there was no significant difference in shortening rate between the control and hypertrophy groups at afterloads of 150, 200 and 250 g/cm2. Endocardial shortening rate at a preload of 25 versus 35 g/cm2 demonstrated a preload dependence in both the control (p less than 0.04) and the hypertrophy group (p less than 0.01). Mid wall shortening rate displayed a preload dependence only in the hypertrophy group (p less than 0.05). It is concluded that end-systolic stress-strain relations are linear in control conditions and in hypertrophy, fiber shortening rate is preload-dependent and, thus, shortening rate-afterload relations currently used to assess myocardial contractility need to be modified to account for these preload effects.

Application of the systolic stiffness concept to assess myocardial function in developing hypertension.

The concept of end-systolic myocardial stiffness permits the quantification of preload effects on fiber shortening and changes in the slope (max Eav) of the end-systolic stress-strain relation, which, if linear, reflect changes in the inotropic state. As an application of this new concept, the end-systolic stress-strain and shortening-afterload relations were evaluated on the basis of data from dogs studied during development of perinephritic hypertension. End-systolic stress-strain relations were linear before and 2 weeks after the induction of hypertension and the end-systolic pressure-diameter relations were not always linear. The shortening-afterload relations obtained directly from raw data points displayed enhanced contractility in the hypertensive state under beta-adrenergic receptor blockade. However, the preload-corrected shortening-afterload relations demonstrated that contractility was unchanged in hypertension. Hypertensive hearts operated at higher preload than normotensive hearts at any afterload levels. This discrepancy between the conventional method without preload-correction and the preload-corrected analysis indicates the importance of preload-correction on shortening-afterload relations in hypertension.

Sympathetic augmentation of cardiac function in developing hypertension in conscious dogs.

To determine the alterations in left ventricular (LV) function and the mechanisms involved that occur during the development of perinephritic hypertension, dogs were instrumented with a miniature LV pressure transducer, aortic and left atrial catheters, and ultrasonic crystals to measure LV diameter in the short and long axes and wall thickness. At 2 wk after initiation of perinephritic hypertension, increases (P less than 0.05) were observed in LV systolic pressure, LV end-diastolic pressure, both short- and long-axis end-diastolic diameters, calculated LV end-diastolic volume, stroke volume, global average LV systolic wall stress, first derivative of LV pressure (LV dP/dt), and ejection fraction, whereas mean velocity of circumferential fiber shortening (Vcf) and rate of change of LV short-axis diameter (LV dD/dt) rose but not significantly. At three levels of matched preload and afterload induced by the administration of graded doses of phenylephrine, Vcf, LV dD/dt, and LV dP/dt increased in hypertension compared with the same levels of preload and afterload before hypertension. When the loading conditions in the normotensive and hypertensive dogs were matched, either after ganglionic blockade or beta-adrenergic blockade, both isovolumic and ejection-phase indexes of LV function remained similar before and after hypertension. Thus we conclude that 1) LV function in intact, conscious dogs with early hypertension is enhanced, and 2) the major mechanism for the increase in LV function involves the sympathetic nervous system.