Intra- and extracellular regulation of activity and processing of legumain by cystatin E/M

Legumain, an asparaginyl endopeptidase, is up-regulated in tumour and tumour-associated cells, and is linked to the processing of cathepsin B, L, and proMMP-2. Although legumain is mainly localized to the endosomal/lysosomal compartments, legumain has been reported to be localized extracellularly in the tumour microenvironment and associated with extracellular matrix and cell surfaces. The most potent endogenous inhibitor of legumain is cystatin E/M, which is a secreted protein synthesised with an export signal. Therefore, we investigated the cellular interplay between legumain and cystatin E/M. As a cell model, HEK293 cells were transfected with legumain cDNA, cystatin E/M cDNA, or both, and over-expressing monoclonal cell lines were selected (termed M38L, M4C, and M3CL, respectively). Secretion of prolegumain from M38L cells was inhibited by treatment with brefeldin A, whereas bafilomycin A1 enhanced the secretion. Cellular processing of prolegumain to the 46 and 36 kDa enzymatically active forms was reduced by treatment with either substance alone. M38L cells showed increased, but M4C cells decreased, cathepsin L processing suggesting a crucial involvement of legumain activity. Furthermore, we observed internalization of cystatin E/M and subsequently decreased intracellular legumain activity. Also, prolegumain was shown to internalize followed by increased intracellular legumain processing and activation. In addition, in M4C cells incomplete processing of the internalized prolegumain was observed, as well as nuclear localized cystatin E/M. Furthermore, auto-activation of secreted prolegumain was inhibited by cystatin E/M, which for the first time shows a regulatory role of cystatin E/M in controlling both intra- and extracellular legumain activity.     

Stochastic population dynamics and life-history variation in marine fish species

Abstract We examined whether differences in life-history characteristics can explain interspecific variation in stochastic population dynamics in nine marine fish species living in the Barents Sea system. After observation errors in population estimates were accounted for, temporal variability in natural mortality rate, annual recruitment, and population growth rate was negatively related to generation time. Mean natural mortality rate, annual recruitment, and population growth rate were lower in long-lived species than in short-lived species. Thus, important species-specific characteristics of the population dynamics were related to the species position along the slow-fast continuum of life-history variation. These relationships were further associated with interspecific differences in ecology: species at the fast end were mainly pelagic, with short generation times and high natural mortality, annual recruitment, and population growth rates, and also showed high temporal variability in those demographic traits. In contrast, species at the slow end were long-lived, deepwater species with low rates and reduced temporal variability in the same demographic traits. These interspecific relationships show that the life-history characteristics of a species can predict basic features of interspecific variation in population dynamical characteristics of marine fish, which should have implications for the choice of harvest strategy to facilitate sustainable yields.     

Amplitude variability and extracellular low-pass filtering of neuronal spikes

The influence of neural morphology and passive electrical parameters on the width and amplitude of extracellular spikes is investigated by combined analytical and numerical investigations of idealized and anatomically reconstructed pyramidal and stellate neuron models. The main results are: 1), All models yield a low-pass filtering effect, that is, a spike-width increase with increasing distance from soma. 2), A neuron's extracellular spike amplitude is seen to be approximately proportional to the sum of the dendritic cross-sectional areas of all dendritic branches connected to the soma. Thus, neurons with many, thick dendrites connected to soma will produce large amplitude spikes, and therefore have the largest radius of visibility. 3), The spike shape and amplitude are found to be dependent on the membrane capacitance and axial resistivity, but not on the membrane resistivity. 4), The spike-amplitude decay with distance r is found to depend on dendritic morphology, and is decaying as 1/rⁿ with 1 ≤ n ≤ 2 close to soma and n ≥ 2 far away.     

Effect of two pyrimidine analogs on accumulation of tubulin in NHIK 3025 cells

Summary Accumulation of tubulin as compared with the accumulation of total cellular protein in human NHIK 3025 cells treated with the sulfone 2-(2-thenyl)sulfonyl-5-bromopyrimidine (NY 4137) and the sulfoxide 2-(2thenyl)sulfinyl-5-bromopyrimidine (NY 4138), two mitotic inhibitors, were investigated by two-parametric flow cytometry. Following a 4 h treatment with NY 4137 tubulin accumulation is inhibited while total protein continues to accumulate. After treatment for 4h with NY 4138 the accumulation of total protein is approximately constant, while the accumulation of tubulin is reduced although not to the same degree as that found for NY 4137-treated cells. In addition, the percentage tubulin SH-groups (6.89 ± 0.14) remaining after treatment of purified rat brain tubulin with NY 4137 or NY 4138 was determined. Treatment with 0.0125 mM NY 4137 reduced the number of tubulin SH-groups detectable with dithiobis benzoate or from 6.89 ± 0.14 before treatment to about 4 after treatment. However, practically all SH-groups of tubulin remain detectable following treatment with the same concentration of NY 4138. From the results described in this report we infer that NY 4137 binds to tubulin SH-groups and that inhibition of tubulin accumulation follows as a secondary effect.     

Hypoxia-Induced Apoptosis in Human Cells with Normal p53 Status and Function, without Any Alteration in the Nuclear Protein Level

We have studied hypoxia-induced inactivation of cells from three established human cell lines with different p53 status. Hypoxia was found to induce apoptosis in cells expressing wild-type p53 (MCF-7 cells), but not in cells where p53 is either mutated (T-47D cells), or abrogated by expression of the HPV18 E6 oncoprotein (NHIK 3025 cells). Apoptosis was demonstrated by DNA fragmentation, using agarose gel electrophoresis of DNA and DNA nick end labeling (TUNEL). We demonstrate that extremely hypoxic conditions (<4 ppm O₂) do not cause any change of expression in the p53 protein level in these three cell lines. In addition, the localization of p53 in MCF-7 cells was found exclusively in the nucleus in only some of the cells both under aerobic and hypoxic conditions. Furthermore, no correlation was found between the p53-expression level and whether or not a cell underwent apoptosis. Flow cytometric TUNEL analysis of MCF-7 cells revealed that initiation of apoptosis occurred in all phases of the cell cycle, although predominantly for cells in S phase. Apoptosis was observed only during a limited time window (i.e., ≈10 to ≈24 h) after the onset of extreme hypoxia. While 66% of the MCF-7 cells lost their ability to form visible colonies following 15 h exposure to extreme hypoxia, only ∼28% were induced to apoptosis, suggesting that ∼38% were inactivated by other death processes. Commitment to apoptotic cell death was observed in MCF-7 cells even for oxygen concentrations as high as 5000 ppm. Our present results indicate that the p53 status in these three tumor cell lines does not have any major influence on cell's survival following exposure to extremely hypoxic conditions, whereas following moderate hypoxia, cells expressing functional p53 enhanced their susceptibility to cell death. Taken together, although these results suggest that functional p53 might play a role in the induction of apoptosis during hypoxia, other factors seem to be equally important.     

An exploratory trial of cyclooxygenase type 2 inhibitor in HIV-1 infection: downregulated immune activation and improved T cell-dependent vaccine responses

Chronic HIV infection is characterized by chronic immune activation and dysfunctional T cells with elevated intracellular cyclic AMP (cAMP), which inhibits the T cell activation capability. cAMP may be induced by prostaglandin E(2) following lipopolysaccharide (LPS)-induced upregulation of cyclooxygenase type 2 (COX-2) in monocytes due to the elevated LPS levels in patients with chronic HIV infection. This hypothesis was tested using celecoxib, a COX-2 inhibitor, for 12 weeks in HIV-infected patients without antiretroviral treatment in a prospective, open, randomized exploratory trial. Thirty-one patients were randomized in the trial; 27 completed the study, including 13 patients on celecoxib. Celecoxib reduced chronic immune activation in terms of CD38 density on CD8(+) T cells (-24%; P = 0.04), IgA levels (P = 0.04), and a combined score for inflammatory markers (P < 0.05). Celecoxib further reduced the inhibitory surface receptor programmed death 1 (PD-1) on CD8(+) T cells (P = 0.01), including PD-1 on the HIV Gag-specific subset (P = 0.02), enhanced the number of CD3(+) CD4(+) CD25(+) CD127(lo/-) Treg or activated cells (P = 0.02), and improved humoral memory recall responses to a T cell-dependent vaccine (P = 0.04). HIV RNA (P = 0.06) and D dimers (P = 0.07) tended to increase in the controls, whereas interleukin-6 (IL-6) possibly decreased in the treatment arm (P = 0.10). In conclusion, celecoxib downmodulated the immune activation related to clinical progression of chronic HIV infection and improved T cell-dependent functions in vivo.     

Uracil-DNA glycosylase in base excision repair and adaptive immunity: species differences between man and mouse

Genomic uracil is a DNA lesion but also an essential key intermediate in adaptive immunity. In B cells, activation-induced cytidine deaminase deaminates cytosine to uracil (U:G mispairs) in Ig genes to initiate antibody maturation. Uracil-DNA glycosylases (UDGs) such as uracil N-glycosylase (UNG), single strand-selective monofunctional uracil-DNA glycosylase 1 (SMUG1), and thymine-DNA glycosylase remove uracil from DNA. Gene-targeted mouse models are extensively used to investigate the role of these enzymes in DNA repair and Ig diversification. However, possible species differences in uracil processing in humans and mice are yet not established. To address this, we analyzed UDG activities and quantities in human and mouse cell lines and in splenic B cells from Ung(+/+) and Ung(-/-) backcrossed mice. Interestingly, human cells displayed ～15-fold higher total uracil excision capacity due to higher levels of UNG. In contrast, SMUG1 activity was ～8-fold higher in mouse cells, constituting ～50% of the total U:G excision activity compared with less than 1% in human cells. In activated B cells, both UNG and SMUG1 activities were at levels comparable with those measured for mouse cell lines. Moreover, SMUG1 activity per cell was not down-regulated after activation. We therefore suggest that SMUG1 may work as a weak backup activity for UNG2 during class switch recombination in Ung(-/-) mice. Our results reveal significant species differences in genomic uracil processing. These findings should be taken into account when mouse models are used in studies of uracil DNA repair and adaptive immunity.     

Power Laws from Linear Neuronal Cable Theory: Power Spectral Densities of the Soma Potential,Soma Membrane Current and Single-Neuron Contribution to the EEG

Power laws, that is, power spectral densities (PSDs) exhibiting behavior for large frequencies f, have been observed both in microscopic (neural membrane potentials and currents) and macroscopic (electroencephalography; EEG) recordings. While complex network behavior has been suggested to be at the root of this phenomenon, we here demonstrate a possible origin of such power laws in the biophysical properties of single neurons described by the standard cable equation. Taking advantage of the analytical tractability of the so called ball and stick neuron model, we derive general expressions for the PSD transfer functions for a set of measures of neuronal activity: the soma membrane current, the current-dipole moment (corresponding to the single-neuron EEG contribution), and the soma membrane potential. These PSD transfer functions relate the PSDs of the respective measurements to the PSDs of the noisy input currents. With homogeneously distributed input currents across the neuronal membrane we find that all PSD transfer functions express asymptotic high-frequency power laws with power-law exponents analytically identified as for the soma membrane current, for the current-dipole moment, and for the soma membrane potential. Comparison with available data suggests that the apparent power laws observed in the high-frequency end of the PSD spectra may stem from uncorrelated current sources which are homogeneously distributed across the neural membranes and themselves exhibit pink () noise distributions. While the PSD noise spectra at low frequencies may be dominated by synaptic noise, our findings suggest that the high-frequency power laws may originate in noise from intrinsic ion channels. The significance of this finding goes beyond neuroscience as it demonstrates how power laws with a wide range of values for the power-law exponent α may arise from a simple, linear partial differential equation.     

Comparison between flow cytometry and image cytometry in ploidy distribution assessments in gynecologic cancer.

The DNA content in 37 tumors from 34 women with gynecological cancer was measured by flow cytometry (FCM) and interactive image cytometry (ICM). Agreement was obtained in 81% of cases as regards ploidy levels, but seven tumors (19%) showed different ploidies. Of these, five were classified as diploid by FCM but either aneuploid (three cases) or polyploid (two cases) by ICM. Two other tumors were aneuploid by ICM but polyploid (one case) and unclassifiable (one case) by FCM. All tumors classified as aneuploid by FCM were also aneuploid by ICM, and all tumors classified diploid by ICM were also diploid by FCM. Of six patients whose tumors were classified as euploid (five diploid and one polyploid) by FCM but classified as aneuploid by ICM, five relapsed, and three of these have died of disease. On the basis of these findings, it is concluded that ICM must be performed in cases classified as diploid by FCM to ensure that small subpopulations of aneuploid tumor cells are not overlooked.