Characterization and differential distribution of the three major human protein kinase C isozymes (PKC alpha, PKC beta, and PKC gamma) of the central nervous system in normal and Alzheimer's disease brains

Brain kinases may play important roles in normal memory as well as in the pathogenesis of neurodegenerative diseases. However, there is scant information on these enzymes in the human brain. For this reason, we characterized the immunohistochemical localization of protein kinase C (PKC) isozymes in human Alzheimer's disease (AD) and non-AD control brains. Using monoclonal antibodies to PKC alpha, PKC beta, and PKC gamma isozymes, we (a) determined the distribution of each PKC isozyme in eight different brain regions (cerebellum, hippocampus, as well as midfrontal, orbital frontal, motor, occipital, parietal, and temporal cortices) from AD and non-AD control brains and found that these patterns were generally similar to those in the rat brain; (b) showed that there were no significant differences in the normal staining intensity of the monoclonal antibodies in AD and non-AD control brain regions except in the hippocampus; (c) observed striking PKC immunoreactivity in globular and linear profiles in the periphery (corona) of AD senile plaques; and (d) demonstrated that PKC alpha immunoreactivity was enhanced in reactive astrocytes associated with senile plaques and other lesions (embolic infarcts) in both AD and non-AD control brains. The data on the normal distribution of each PKC isozyme were corroborated in quantitative studies of PKC alpha, PKC beta, and PKC gamma protein levels in human postmortem brain regions by Western blotting using our antibodies. We conclude that these three major PKC isozymes can be analyzed directly in postmortem human brain, which is an important first step in understanding the potential role that abnormal phosphorylation might play in the pathogenesis of AD.     

Preliminary observations on polar body extrusion and pronuclear formation in human oocytes using time-lapse video cinematography

In this study, we have used time-lapse video cinematography to study fertilization in 50 human oocytes that had undergone intracytoplasmic sperm injection (ICSI). Time-lapse recording commenced shortly after ICSI and proceeded for 17-20 h. Oocytes were cultured in an environmental chamber which was maintained under standard culture conditions. Overall, 38 oocytes (76%) were fertilized normally, and the fertilization rate and embryo quality were not significantly different from 487 sibling oocytes cultured in a conventional incubator. Normal fertilization followed a defined course of events, although the timing of these events varied markedly between oocytes. In 35 of the 38 fertilized oocytes (92%), there were circular waves of granulation within the ooplasm which had a periodicity of 20-53 min. The sperm head decondensed during this granulation phase. The second polar body was then extruded, and this was followed by the central formation of the male pronucleus. The female pronucleus formed in the cytoplasm adjacent to the second polar body at the same time as, or slightly after, the male pronucleus, and was subsequently drawn towards the male pronucleus until the two abutted. Both pronuclei then increased in size, the nucleoli moved around within the pronuclei and some nucleoli coalesced. During pronuclear growth, the organelles contracted from the cortex towards the centre of the oocyte, leaving a clear cortical zone. The oocyte decreased in diameter from 112 to 106 microm (P < 0.0001) during the course of the observation period. The female pronucleus was significantly smaller in diameter than the male pronucleus (24.1 and 22.4 microm respectively, P = 0.008) and contained fewer nucleoli (4.2 and 7.0 respectively, P < 0.0001). After time-lapse recording, oocytes were cultured for 48 h prior to embryo transfer or cryopreservation. Embryo quality was related to fertilization events and periodicity of the cytoplasmic wave, and it was found that good quality embryos arose from oocytes that had more uniform timing from injection to pronuclear abuttal and tended to have a longer cytoplasmic wave. In conclusion, we have shown that time-lapse video cinematography is an excellent tool for studying fertilization and early embryo development, and have demonstrated that human fertilization comprises numerous complex dynamic events.      

Lymphocyte proliferation in response to acute heavy resistance exercise in women: influence of muscle strength and total work

Little is understood about the immune responses to heavy resistance exercise. The purpose of this investigation was to determine the influence of physical strength and the ability to do more total work on lymphocyte proliferation after an acute bout of heavy resistance exercise. A group of 50 healthy but non-strength trained women were recruited for the study and tested for their one repetition maximum (i.e. 1 RM or maximal mass lifted once). From the normal distribution of strength the top and bottom 8 women [mean age 22.5 (SD 3.1) years] were asked to volunteer to define our two groups (i.e. high strength and low strength). The two groups were significantly different (P<0.05) in 1 RM squat strength [low strength 39.9 (SD 4.6) kg, 0.65 (SD 0.08) kg·kg body mass^–1 and high strength 72.2 (SD 10.7) kg, 1.1 (SD 0.12) kg·kg body mass^–1] but were not significantly different in body mass, age, activity levels, and menstrual status (all in same phase). Each performed a resistance exercise protocol consisting of six sets of 10 RM squats with 2 min rest between the sets. The 10 RM loads and total work were significantly greater in the high strength group than in the low strength group. Blood samples were obtained pre-exercise and immediately post-exercise for test for lactate (significant increase with exercise) and cortisol (no changes) concentrations with no differences noted between groups. Immunological assays on the blood samples determined the incorporation of tritiated thymidine by lymphocytes in responses to concanavalin A (ConA), phytohemagglutinin (PHA), and pokeweed mitogen (PWM). Following the squat exercise, there was a significant decrease in lymphocyte responsiveness to PWM in the high strength but not in the low strength group for both total proliferation and proliferation adjusted per B or T cell. On the other hand, lymphocytes from the low strength group proliferated to a significantly greater extent (adjusted per T cell) in response to ConA and PHA. These data indicate that the heavy resistance exercise protocol reduced the lymphocyte proliferative responses only in the stronger group of subjects. This effect may have been due to the high absolute total work and the greater exercise stress created by the resistance exercise protocol in the high strength group. Therefore, individuals performing at the same relative exercise intensity (i.e. 10 RM) in a resistance exercise protocol may have different immune responses stemming from differences in absolute total work performance. Electronic Publication     

Dual-contrast echo planar imaging with keyhole: application to dynamic contrast-enhanced perfusion studies

A new EPI-based method is presented which features optimized sampling of k-space enabling the integrated acquisition of two gradient echo images. The first of these images is predominantly T1 weighted and the second is T*2 weighted. The new method combines echo sharing of sparsely acquired high spatial frequency components with the keyhole technique and half-Fourier image reconstruction. The feasibility of acquiring high spatial and temporal resolution in vivo images for perfusion mapping is demonstrated. In contrast to most current perfusion methods, which acquire the T1- and T*2-weighted images in separate acquisitions, the need for image co-registration here is obviated since both sets of images are EPI-based and are acquired within the same measurement.     

Levels and specificity of antibody in bronchoalveolar lavage (BAL) and serum in an animal model of trimellitic anhydride-induced lung injury

A study was undertaken to characterize the antibody response in rats exposed to trimellitic anhydride (TMA) by inhalation. Total antibody levels directed to trimellitic rat serum albumin (TM-RSA) from TMA-exposed rats were assayed by an ammonium sulfate technique. Total antibody levels in bronchoalveolar lavage (BAL) and the matched serum were compared by correction for the albumin content of each. An ELISA was developed to detect IgG, IgA, and IgM directed toward TM-RSA in BAL and serum and to compare class-specific antibody levels in BAL and serum by normalizing for albumin content. The specificity of the rat IgG response was determined by ELISA inhibition with TM-RSA and TM-human serum albumin (TM-HSA) and compared with reciprocal inhibition studies with serum from TMA-exposed workers. The levels of total antibody in BAL were three to 15 times greater than the levels found in the matched serum pair. IgG, IgA, and IgM antibodies were detected in the BAL and the serum of TMA-exposed rats but not in control rats. In each of the four rats tested, all antibody classes were present in equal or greater amounts in the BAL than in the serum. Complete inhibition of the rat IgG binding in ELISA was observed when TM-RSA or TM-HSA were added as inhibitors. Human IgG was inhibited in ELISA only by TM-HSA. In an animal model of human lung disease, the levels of total antibody as well as class-specific antibodies directed against TM-RSA were greater in BAL than in serum.(ABSTRACT TRUNCATED AT 250 WORDS)     

Avian tibial dyschondroplasia. III. Electron probe analysis.

Tibial dyschondroplastic (TD) lesions and their associated growth plates, obtained from chickens, were prepared by freeze-drying and embedding in an anhydrous epoxy resin. Quantitative electron probe analysis was performed on dry, unstained sections. Levels of Na, Mg, P, S, Cl, K, and Ca were determined in cytoplasm (endoplasmic reticulum), mitochondria, and extracellular matrix of the proliferative, prehypertrophic, and early hypertrophic zones of the growth plate and in the proximal, mid, and distal regions of the lesion. A zone of calcification in the growth plate was absent. The concentration of elements in all regions of the TD growth plate was the same as found in an earlier study for normal growth plate. The cytoplasm of proximal lesion chondrocytes was similar to that of early hypertrophic chondrocytes. However, in the remainder of the lesion there was a progressive increase in cellular Na, S, Cl, and Ca and a progressive loss of P. In matrix, there was less S and K than expected in all regions of growth plate and lesion, except in the proliferating zone. Also, in matrix of the distal lesion there was less Na and Cl. The levels of Na, S, Cl, and K in matrix may have been lowered by their adsorption into the condensed masses of dead cells. Mitochondria acquire only half as much Ca and P as normal and release it earlier than usual (ie, early prehypertrophic cells, rather than chondrocytes of the lower hypertrophic zone). There were no granules in mitochondria of the cells at all levels of the lesion, even though anhydrous methods were used. The first sign of the disease appears in the matrix of the growth plate, where it seems that S and K are in abnormally low amounts. Although there are sufficient levels of Ca and P present, the matrix does not calcify. The cartilage remains avascular, and the cells appear to be dying. The event that triggers the chondrocytes of the growth plate to form an abnormal uncalcified matrix is not known.     

Phenotypically distinct subsets of CD4+ T cells induce or protect from chronic intestinal inflammation in C. B-17 scid mice

CD4⁺ T cells in the mouse can be subdivided into two fractionsbased on the level of expression of the CD45RB determinant.Previous studies have shown that these subsets are functionallydistinct. We have further characterized the properties of thesesubpopulations in vivo by injecting them into C. B-17 scid mice.The animals restored with the CD45RB^highCD4⁺ T cell populationdeveloped a lethal wasting disease with severe mononuclear cellinfiltrates into the colon and elevated levels of IFN- mRNA.In contrast, animals restored with the reciprocal CD45RB^lowsubset or with unfractionated CD4⁺ T cells did not develop thewasting or colitis. Importantly, the co-transfer of the CD45RB^lowpopulation with the CD45RB^high population prevented the wastingdisease and colitis. These data indicate that important regulatoryinteractions occur between the CD45RB^high and CD45RB^lowCD4⁺T cell subsets and that disruption of this mechanism has fatalconsequences.     

On the oxygenation-dependent (129)Xe T (1) in blood

The spin-lattice relaxation time, T(1), of hyperpolarized (129)Xe in blood is sensitive to blood oxygenation. In particular, it has been shown that (129)Xe T(1) is shorter in venous blood than in arterial blood. We have studied the T(1) of hyperpolarized (129)Xe dissolved in human blood as a function of blood oxygenation level, sO(2), in the physiological oxygenation range. We show that the (129)Xe relaxation rate, T(1)(-1), varies in a nonlinear fashion as a function of sO(2). This finding suggests that direct interaction of xenon with the paramagnetic heme group of deoxyhemoglobin is not the dominant oxygenation-dependent relaxation mechanism for (129)Xe in blood. These results corroborate the idea that the oxygenation-dependence of (129)Xe T(1) is determined by conformational changes of hemoglobin induced by oxygen binding.