Nanoparticle-mediated catalase delivery protects human neurons from oxidative stress.

Several neurodegenerative diseases and brain injury involve reactive oxygen species and implicate oxidative stress in disease mechanisms. Hydrogen peroxide (H2O2) formation due to mitochondrial superoxide leakage perpetuates oxidative stress in neuronal injury. Catalase, an H2O2-degrading enzyme, thus remains an important antioxidant therapy target. However, catalase therapy is restricted by its labile nature and inadequate delivery. Here, a nanotechnology approach was evaluated using catalase-loaded, poly(lactic co-glycolic acid) nanoparticles (NPs) in human neuronal protection against oxidative damage. This study showed highly efficient catalase encapsulation capable of retaining ~99% enzymatic activity. NPs released catalase rapidly, and antioxidant activity was sustained for over a month. NP uptake in human neurons was rapid and nontoxic. Although human neurons were highly sensitive to H2O2, NP-mediated catalase delivery successfully protected cultured neurons from H2O2-induced oxidative stress. Catalase-loaded NPs significantly reduced H2O2-induced protein oxidation, DNA damage, mitochondrial membrane transition pore opening and loss of cell membrane integrity and restored neuronal morphology, neurite network and microtubule-associated protein-2 levels. Further, catalase-loaded NPs improved neuronal recovery from H2O2 pre-exposure better than free catalase, suggesting possible applications in ameliorating stroke-relevant oxidative stress. Brain targeting of catalase-loaded NPs may find wide therapeutic applications for oxidative stress-associated acute and chronic neurodegenerative disorders.

Sudden cardiac death in the young: a 1-year post-mortem analysis in the Republic of Ireland.

AIMS: A retrospective post-mortem analysis was undertaken to determine the aetiology of sudden cardiac death (SCD) amongst individuals aged

p53 localizes to the centrosomes and spindles of mitotic cells in the embryonic chick epiblast, human cell lines, and a human primary culture: An immunofluorescence study.

Immunofluorescent staining of mitotic centrosomes and spindles by anti-p53 antibodies was observed in the embryonic chick epiblast by epifluorescence microscopy and in three human cancer cell lines, an SV40-immortalized cell line, and a normal human fibroblast culture by confocal microscopy. In the chick epiblast, the centrosomes stained from early prophase through to the formation of the G1 nuclei and the spindle fibers stained from prophase through to telophase. In the human cells, the staining was observed from late prophase to telophase. The epiblast was stained by the anti-p53 antibodies DO-1, Ab-6, and Bp53-12. The human cells were also stained by these antibodies as well as by other anti-p53 antibodies. Preabsorption of DO-1 and Bp53-12 with purified tubulin did not diminish the immunostaining, showing that the antibodies were not reacting with tubulin in the mitotic centrosomes and spindles. The immunostaining in the chick epiblast was very clearly localized to the mitotic centrosomes and spindles, revealing a cytoplasmic location for p53 during mitosis and accounting for earlier reports of an association between p53, tubulin, and centrosomes. The localization of p53 to the spindle supports an involvement of p53 in spindle function.

Hox-type and non-Hox homeobox gene sequences in genomic DNA of the sea urchin Holopneustes purpurescens.

As a preliminary step in an analysis of Hox gene expression and radial body plan specification in sea urchin development, we amplified partial homeobox sequences in H. purpurescens by PCR using degenerate primers. The primers, HoxE and HoxF (Pendleton et al., 1993), spanned a highly conserved region of 82 nucleotides encompassing amino acids 21-47 of the homeodomain. Seven Hox-type homeobox sequences and two non-Hox homeobox sequences were identified. The seven Hox-type sequences were placed provisionally in Hox paralogous groups, one in paralogous group 3, three in paralogous groups 6-8 and three in paralogous groups 9 13. The non-Hox sequences had similarities with Xlox and Gbx homeobox genes.

Donor site morbidity of the anterior iliac crest following cancellous bone harvest.

Anterior iliac crest donor site morbidity was analysed retrospectively at 154 sites in 137 patients who had undergone secondary bone grafting of the cleft alveolus. The applied surgical anatomy and operative technique utilizing a medially based osteoplastic flap are described. The results show a low incidence of donor site morbidity.

An analysis of the growth of the retinal cell population in embryonic chicks yielding proliferative ratios, numbers of proliferative and non-proliferative cells and cell-cycle times for successive generations of cell cycles.

Growth curves of the retinal cell population of embryonic chicks were fitted by a branching-process model of cell population growth, thereby estimating the proliferative ratios and mean cell-cycle times of the generations of cell cycles that underlie retinal growth. The proliferative ratio determines the proportion of cells that divides in the next generation, so the numbers of proliferative and non-proliferative cells in each generation of cell cycles were obtained. The mean cell-cycle times determine the times over which the generations are extant. Assuming growth starts from one cell in generation 0, the proliferative cells reach 3.6 x 10(6) and the non-proliferative cells reach 1.1 x 10(6) by generation 23. The next four generations increase the proliferative cell numbers to 13.9 x 10(6) and produce 20.1 x 10(6) non-proliferative cells. In the next five generations in the end phase of growth, non-proliferative cells are produced in large numbers at an average of 13.9 x 10(6) cells per generation as the retinal lineages are completed. The retinal cell population reaches a maximum estimated here at 98.2 x 10(6) cells. The mean cell-cycle time estimates range between 6.8 and 10.1 h in generations before the end phase of growth and between 10.6 and 17.2 h in generations in the end phase. The retinal cell population growth is limited by the depletion of the proliferative cell population that the production of non-proliferative cells entails. The proliferative ratios and the cell-cycle-time distribution parameters are the likely determinants of retinal growth rates. The results are discussed in relation to other results of spatial and temporal patterns of the cessation of cell cycling in the embryonic chick retina.

Spatial patterns of cells in dividing epithelia.

The spatial patterns of cell boundaries in a view of the apical surface of a dividing epithelium are explored by constructing a hypothetical cell pattern of an epithelium of dividing cells. The two elements specified in the hypothetical pattern are the orientation of division planes and the separation between the division planes in neighbouring cells. The orientations of division planes in one generation are all the same but are orthogonal to those in the preceding generation. The division-plane orientations follow in an orthogonal succession, as happens in early embryos. The division planes in neighbouring cells are offset. The contractions of division planes that would occur during cytokinesis distort existing boundaries creating various types of cell shapes. The patterns generated resemble cell patterns found in life. The hypothetical pattern is regenerative and shows how epithelial cell patterns where cells divide might arise. It has enabled the putative identification of sister cells and first cousins in the embryonic chick chorion.

Heat shock affects cell cycling in the neural plate of cultured rat embryos: a flow cytometric study.

The effects of heat shock on cell cycling in the mammalian neuroectoderm were determined by applying heat shocks to cultured rat embryos at the neural plate stage, as part of a study on the teratogenic effects of heat shock on neural development. The heat shocks had been characterized previously (Walsh et al.: Teratology 36:181-191, 1987) with respect to their effects on the gross morphological development of the rat embryos. The effects on cell cycling were observed in DNA histograms of neural plate cells recorded in a flow cytometer after staining with DAPI. The mild heat shock (42 degrees C for 10 min) arrested cells at entry to S phase. The teratogenic heat shock (43 degrees C for 7.5 min) arrested cells at entry to S phase also but for a longer time and inhibited cycling through S phase. After each arrest, a synchronized peak of cells later entered S phase and progressed through the cycle. The induced-thermotolerance heat shock, which was the mild heat shock followed after an interval by the teratogenic heat shock, showed that pre-treatment with the mild heat shock reduced the magnitude of the response to the teratogenic heat shock. The cell-cycle inhibitor ICRF 159 showed the effects on cycling rates of the heat-shock treatments. The arrest of cells at entry to S phase by heat shock may function to prevent cells entering DNA synthesis under non-optimal conditions. We report estimates of proportions of non-proliferative cells in the neural plate of the rat embryos.

Division rules for polygonal cells.

A number of fascinating mathematical problems concerning the division of two-dimensional space are formulated from questions about the planes of cell division in embryonic epithelia. Their solution aids in the quantitative description of cellular arrangement in epithelia. Cells, considered as polygons, site their division line according to stochastic rules, eventually forming a tessellation of the plane. The equilibrium distributions for the resulting mix of polygonal types are explored for a range of stochastic rules. We find surprising links with some classical distributions from the theory of probability.

Determination of proliferative parameters from growth curves.

Data on total cell numbers and proportions of non-proliferative cells during the development of a tissue can be obtained by experiment. This paper shows, via a simple mathematical model, that data collected over a period of time determine the time profile of two proliferative parameters. These are the rate per proliferative cell per day at which cells divide and the average number of proliferative daughters which result from the division of a proliferative cell. One dataset alone does not provide sufficient information to determine either time profile. The findings are applicable where there is no significant cell death during the growth period. The mathematical model is fitted to data from chick neural retina.

Cell population dynamics during the differentiative phase of tissue development.

The dynamics of a cell population whose numbers are growing exponentially have been described well by a mathematical model based on the theory of age-dependent branching processes. Such a model, however, does not cover the period following exponential growth when cell differentiation curtails population size. This paper offers an extension to the branching process model to remedy this deficiency. The extended model is ideal for describing embryonic growth; its use is illustrated with data from embryonic retina. The model offers a better computational framework for the interpretation of a variety of data (growth curves of cell numbers, DNA histograms, thymidine labelling indices, FLM curves, BUdR-labelled mitoses curves) because age-distributions can be calculated at any stage of development, not just during exponential growth. Proportions of cells in the various phases of the cell cycle can be computed as growth slows. Such calculations show the gradual transition from a population dominated by cells which are young with respect to cell cycle age to one dominated by those which are old, and the effects such biases have on the proportions of cells in each phase.

Estimation of nonproliferating cells in the neural retina of embryonic chicks by flow cytometry.

Nonproliferating cells (N cells) in the neural retina of embryonic chicks were estimated after isolating them in the 2C peak of a DNA distribution by exposure to the cell-cycle inhibitor ICRF 159. ICRF 159 inhibits cell division but not DNA synthesis, so proliferating cells can leave the 2C peak but not reenter it. Cells left in the 2C peak after exposure to ICRF 159 were assumed to be N cells. The effectiveness of ICRF 159 in inhibiting cell division but not inhibiting DNA synthesis was demonstrated in neural retinae from stage 17 embryos which showed no evidence of a 2C peak after 6-h exposure to ICRF 159 and which were thus shown to have no N cells. A test to detect escape from the cell division block in older embryos with an N cell population in the neural retina showed some escape after longer exposures to ICRF 159. The escape was suppressed by a second dose of ICRF 159, given some hours after the first.

Pattern of DNA synthesis and its effect on the classification of cells by flow cytometry.

A flow cytometer produces a DNA distribution which contains information about the proportions of cells in various phases of the cell cycle. In this report we show that the form assumed for the rate of DNA synthesis in a cell plays an important part in the estimation of those proportions. We compare three forms for the rate of DNA synthesis, one of which is derived from knowledge of the mechanism of DNA replication, and find that they give substantially different estimates of the proportions of cells in the G1 and G2 + M phases.

A growth curve of cell numbers in the neural retina of embryonic chicks.

The growth of cell numbers in a normal embryonic population of neural retinal cells is described. The numbers were estimated from a time shortly after the neural retina first becomes recognizable to a time when numbers of retinal cells have become steady. Cell numbers were estimated in preparations of an entire neural retina dispersed into a suspension of single nuclei which were then counted in a Coulter counter. The growth curve of the ln numbers of cells has three phases of growth: an exponential phase during which there is steady-state exponential growth, a differentiative phase during which cell proliferation ceases and an end phase when no further change in cell numbers can be detected. The variances of the ln numbers of cells were highest during the exponential phase. The variances decreased during the differentiative phase and were at their lowest during the end phase. For variances to decrease requires mechanisms which control the final numbers of cells in the neural retina very precisely. The implications of mechanisms which operate by controlling cell lineages are explored.

An afoveate area centralis in the chick retina.

A specialization has been found in the nasal retina of late embryonic and newly hatched chicks of the domestic fowl. In whole mounts of the retina, there is a central node about 2 mm from the dorsal end of the optic disc and a set of structures radiating out from this node. This complex has been named the aster. Sections of the retina show that the structures which form the aster lie in the inner nuclear layer. The specialization has been classed as an afoveate area centralis.

Salter-Harris type-III fracture of the medial femoral condyle occurring in the adolescent athlete.

This report adds six Salter-Harris type-III fractures of the medial femoral condyle to the fourteen such fractures that have been previously reported in the English literature. The injury results from a valgus force applied to the knee. As the fracture may reduce spontaneously, radiographs may not be diagnostic and the fracture may be unrecognized or be mistaken for disruption of the medial collateral ligament. If a fracture is suspected, a cross-table lateral radiograph should be made in an attempt to detect fat within the joint fluid and confirm the existence of an intra-articular fracture. An oblique, tunnel (notch), or over-penetrated radiograph may demonstrate the fracture. However, if it fails to do so, radiographs made with valgus stress applied to the knee while the patient is under general anesthesia may be necessary. In our six patients, satisfactory healing of the fracture occurred after either spontaneous or manipulative reduction and subsequent non-weight-bearing immobilization in cylinder cast. Minimum femoral shortening developed in two patients, late anterior cruciate laxity was demonstrable in two patients, and one patient required arthrotomy for removal of an osteochondral fracture fragment.

Random segregation of sister chromatids in developing chick retinal cells demonstrated in vivo using the fluorescence plus Giemsa technique.

Experiments were designed to test whether nonrandom segregation of sister chromatids at mitosis has a role in the production of cell diversity during embryogenesis, Segregation was examined in vivo in retinal cells from embryonic chicks. Chromatids were labelled with bromouracil and stained by the fluorescence plus Giemsa technique. No evidence of nonrandom segregation was observed in a frequency distribution of pairs of bifilarly labelled sister chromatids at the third metaphase after the start of labeling. Nor was there evidence that chromatids from homologous chromosomes segregated nonrandomly. Nonrandom segregation is probab;y not a mechanism for cell diversification.

The growth of the chick retina after hatching.

In autoradiographs of the retina from two-week old chicks injected with tritiated thymidine during the first week after hatching, the labelled cells were found mainly in the extreme periphery of the retina and the ciliary epithelium of the pars plana. The pattern of labelling in the extreme periphery resembles that observed (Fujita and Horii, '63) in retinae exposed to tritiated thymidine during embryonic development. The mechanisms underlying the histogenesis of retinal cells during growth at the extreme periphery in young stages may be the same as those which operate during embryonic devolopment.