Anti-mouse GPI-PLD antisera highlight structural differences between murine and bovine GPI-PLDs

Despite its well characterised biochemistry, the physiological role of glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) is unknown. Most of the previous studies investigating the distribution of GPI-PLD have focused on the human and bovine forms of the enzyme. Studies on mouse GPI-PLD are rare, partly due to the lack of a specific anti-mouse GPI-PLD antibody, but also due to the apparent low reactivity of existing antibodies to rodent GPI-PLDs. Here we describe the isolation of a mouse liver cDNA, the construction and expression of a recombinant enzyme and the generation of an affinity-purified rabbit anti-mouse GPI-PLD antiserum. The antibody shows good reactivity to partially purified murine and purified bovine GPI-PLD. In contrast, a rat anti-bovine GPI-PLD antibody shows no reactivity with the mouse enzyme and the two antibodies recognise different proteolytic fragments of the bovine enzyme. Comparison between the rodent, bovine and human enzymes indicates that small changes in the amino acid sequence of a short peptide in the mouse and bovine GPI-PLDs may contribute to the different reactivities of the two antisera. We discuss the implications of these results and stress the importance of antibody selection while investigating GPI-PLD in the mouse.     

Altered mechanisms of sympathetic activation during rhythmic forearm exercise in heart failure

In congestive heart failure (CHF), themechanisms of exercise-induced sympathoexcitation are poorly defined.We compared the responses of sympathetic nerve activity directed tomuscle (MSNA) and to skin (SSNA, peroneal microneurography) duringrhythmic handgrip (RHG) at 25% of maximal voluntary contraction andduring posthandgrip circulatory arrest (PHG-CA) in CHF patients with those of an age-matched control group. During RHG, the CHF patients fatigued prematurely. At end exercise, the increase in MSNA was similarin both groups (CHF patients, n = 12;controls, n = 10). However, duringPHG-CA, in the controls MSNA returned to baseline, whereas it remainedelevated in CHF patients (P < 0.05).Similarly, at end exercise, the increase in SSNA was comparable in bothgroups (CHF patients, n = 11;controls, n = 12), whereas SSNAremained elevated during PHG-CA in CHF patients but not in the controls (P < 0.05). In a separate controlgroup (n = 6), even high-intensity static handgrip was not accompanied by sustained elevation of SSNAduring PHG-CA. ³¹P-nuclear magneticresonance spectroscopy during RHG demonstrated significant muscleacidosis and accumulation of inorganic phosphate in CHF patients(n = 7) but not in controls(n = 9). We conclude that in CHFpatients rhythmic forearm exercise leads to premature fatigue andaccumulation of muscle metabolites. The prominent PHG-CA response ofMSNA and SSNA in CHF patients suggests activation of the musclemetaboreflex. Because, in contrast to controls, in CHF patients bothMSNA and SSNA appear to be under muscle metaboreflex control, themechanisms and distribution of sympathetic outflow during exerciseappear to be different from normal.

     

A gimbal-mounted pressurization chamber for macroscopic and microscopic assessment of ocular tissues

The biomechanical model of glaucoma considers intraocular pressure-related stress and resultant strain on load bearing connective tissues of the optic nerve and surrounding peripapillary sclera as one major causative influence that effects cellular, vascular, and axonal components of the optic nerve. By this reasoning, the quantification of variations in the microstructural architecture and macromechanical response of scleral shells in glaucomatous compared to healthy populations provides an insight into any variations that exist between patient populations. While scleral shells have been tested mechanically in planar and pressure-inflation scenarios the link between the macroscopic biomechanical response and the underlying microstructure has not been determined to date. A potential roadblock to determining how the microstructure changes based on pressure is the ability to mount the spherical scleral shells in a method that does not induce unwanted stresses to the samples (for instance, in the flattening of the spherical specimens), and then capturing macroscopic and microscopic changes under pressure. Often what is done is a macroscopic test followed by sample fixation and then imaging to determine microstructural organization. We introduce a novel device and method, which allows spherical samples to be pressurized and macroscopic and microstructural behavior quantified on fully hydrated ocular specimens. The samples are pressurized and a series of markers on the surface of the sclera imaged from several different perspectives and reconstructed between pressure points to allow for mapping of nonhomogenous strain. Pictures are taken from different perspectives through the use of mounting the pressurization scheme in a gimbal that allows for positioning the sample in several different spherical coordinate system configurations. This ability to move the sclera in space about the center of the globe, coupled with an upright multiphoton microscope, allows for collecting collagen, and elastin signal in a rapid automated fashion so the entire globe can be imaged.