In search of an effective cell disruption method to isolate intact mitochondria from Chinese hamster ovary cells

An efficient isolation of mitochondria from cells under physiological conditions is crucial for many studies in life sciences but still challenging in many cases such as in metabolic characterization of mitochondria. In this work, four methods for the disruption of Chinese hamster ovary cells were evaluated regarding their influence on mitochondrial integrity and yield. After cell disruption, mitochondria released from cells were separated from the remaining cell homogenate by differential centrifugation. Sonication was shown to be a rapid and sensitive isolation method. Yields of 14.0 ± 0.3 mg raw mitochondrial protein per 10⁸ cells were obtained. The mitochondria were morphologically intact, with membrane integrities of 67% (outer membrane) to 94% (inner membrane). Compared with the methods using Dounce homogenization, digitonin permeabilization, or electroporation for cell disruption the ultrasound method provided the highest yield of isolated mitochondria. Furthermore, this method is rapid (≈ 45 s for disruption), more robust than Dounce homogenization regarding their influence on mitochondrial integrity and especially suitable for preparing a relatively large amount of mitochondria. The results of this work can be helpful for quantitative and dynamic studies of molecular processes related to mitochondria under physiological conditions for many questions in both biomedicine and biotechnology.     

Model-Based Analysis of Mechanisms Responsible for Sleep-Induced Carbon Dioxide Differences

This work describes a comprehensive mathematical model of the human respiratory control system which incorporates the central mechanisms for predicting sleep-induced changes in chemical regulation of ventilation. The model integrates four individual compartments for gas storage and exchange, namely alveolar air, pulmonary blood, tissue capillary blood, body tissues, and gas transport between them. An essential mechanism in the carbon dioxide transport is its dissociation into bicarbonate and acid, where a buffering mechanism through hemoglobin is used to prevent harmfully low pH levels. In the current model, we assume high oxygen levels and consider intracellular hydrogen ion concentration as the principal respiratory control variable. The resulting system of delayed differential equations is solved numerically. With an appropriate choice of key parameters, such as velocity of blood flow and gain of a non-linear controller function, the model provides steady-state results consistent with our experimental observations measured in subjects across sleep onset. Dynamic predictions from the model give new insights into the behaviour of the system in subjects with different buffering capacities and suggest novel hypotheses for future experimental and clinical studies.     

A Mathematical Model for the Regulation of Tumor Dormancy Based on Enzyme Kinetics

In this paper we present a two-compartment model for tumor dormancy based on an idea of Zetter [1998, Ann. Rev. Med. 49, 407–422] to wit: The vascularization of a secondary (daughter) tumor can be suppressed by an inhibitor originating from a larger primary (mother) tumor. We apply this idea at the avascular level to develop a model for the remote suppression of secondary avascular tumors via the secretion of primary avascular tumor inhibitors. The model gives good agreement with the observations of [De Giorgi et al., 2003, Derm. Surgery 29, 664–667]. These authors reported on the emergence of a polypoid melanoma at a site remote from a primary polypoid melanoma after excision of the latter. The authors observed no recurrence of the melanoma at the primary site, but did observe secondary tumors at secondary sites 5–7 cm from the primary site within a period of 1 month after the excision of the primary site. We attempt to provide a reasonable biochemical/cell biological model for this phenomenon. We show that when the tumors are sufficiently remote, the primary tumor will not influence the secondary tumor while, if they are too close together, the primary tumor can effectively prevent the growth of the secondary tumor, even after it is removed. It should be possible to use the model as the basis for a testable hypothesis.     

Estimation in regression models with externally estimated parameters

In many regression applications, some of the model parameters are estimated from separate data sources. Typically, these estimates are plugged into the regression model and the remainder of the parameters is estimated from the primary data source. This situation arises frequently in compartment modeling when there is an external input function to the system. This paper provides asymptotic and bootstrap-based approaches for accounting for all sources of variability when computing standard errors for estimated regression model parameters. Examples and simulations are provided to motivate and illustrate the ideas.     

Phosphorus concentration in sediment, water and tissues of␣three submerged macrophytes of Myall Lake, Australia

Phosphorus content in sediment, water and tissues of three macrophyte species growing in Myall Lake, Australia were studied from January to November, 2004. The sites investigated were North–West (NW), North–East (NE), South–West (SW) bays and Central deep area of the lake (CL). The objective of this study was to investigate how total phosphorus (TP) in plant tissues relate to phosphorus pools and the role played by the aquatic macrophyte species under investigation in phosphorus recycling in the lake. Of the four investigated sites of the lake, TP in plant tissues were significantly higher in North–West and South–West bays compared to the rest. Najas marina had significantly higher TP content (e.g., 1.55 and 1.44 mg/g dw.; P < 0.05) for NW and SW respectively, than the other two species. N. marina is a rooted macrophyte while charophytes (C. fibrosa and Nitella hyalina) are pseudo-rooted macrophytes. Total phosphorus in the sediment and water column were significantly higher in Central deep area of the lake compared to the other three bays (P < 0.05, n 5). Soluble reactive phosphorus (SRP) and total dissolved phosphorus (TDP) in sediment pore water correlated significantly with phosphorus content in the tissue of N. marina ( ; ) as well as TP in sediment (␣ and ). Using the two-compartmental uptake model, it was observed that, sediment was the main compartment through which Ni. hyalina obtained phosphorus while for the other two species, water column was the uptake route for the phosphorus. These correlations suggest that, water column and sediments are important pathways for phosphorus uptake in plants.     

Proliferation of endothelial cells in neoangiogenesis of human cortical glioblastoma

In 1982 Vilanova et al. quantitatively described the neovessel area in glioblastoma and suggested zonal differences in vessel surface area. In this study we investigated specific cell proliferation and angiogenic patterns in the vessel compartment of cortical glioblastoma. We used Ki67, CD34 and SMA double immunohistochemical staining to quantitate vascular patterns and cell specific proliferation and presented glioblastoma with several parameters of angiogenesis. Endothelial cell proliferation was higher in complex and bizzare neovessels than in the simple and sprouting glioblastoma neovessels. There was a higher frequency of sprouting simple vessels in close proximity to the palisade and a higher frequency of bizzare vessels in the microzone distant to the palisade. Quantitatively presented for the first time, the neovessel proliferation patterns support cortical glioblastoma compartmentalization. The data obtained are relevant to medical doctors using neoangiogenesis in diagnosis, prognosis and therapy of neoplasia. The results obtained in 15 patients call for further investigation of endothelial cell/pericyte relationships and glioblastoma compartmentalization.     

How does spatio-temporal disturbance influence species diversity in a hierarchical competitive system? Prospective order of species coexistence and extinction

To address how habitat destruction and hierarchical competition among species affect the spatio-temporal dynamics of a multi-species community, we present a compartment model in which multiple species undergo dispersal and competitive interactions in a patchy habitat arranged in a two-dimensional lattice. We assume that disturbances are periodically imposed on some parts of the lattice in a block, followed by a period free of disturbance. For convenience, species are ranked in order of competitive ability. We further assume that the intrinsic growth rate of species i, _i , and the dispersal ability, D _i , increase in decreasing order of rank. Our model can analytically determine the exact number of surviving species when disturbance is absent. In the presence of disturbance, we numerically examine how spatio-temporal changes in environmental heterogeneity affect species coexistence and extinction, for the case in which the value of _i /D _i monotonically increases or decreases with rank. The results demonstrate that (1) when the interspecific competition is smaller than the intraspecific competition, we can provide predictions on the prospective order of species to be driven extinct and the order of potential species to revive with increasing extents of disturbance; (2) when the interspecific competition is stronger than intraspecific competition, a small difference in the disturbance level can lead to drastic changes in the species composition, their densities and the order of species extinction. In addition, comparison with other similar models reveals that differences in species interaction in local population dynamics critically affect the disturbance-mediated species diversity.     

Non-canonical Maturation of Two Papain-family Proteases in Toxoplasma gondii

Proteases regulate key events during infection by the pervasive intracellular parasite Toxoplasma gondii. Understanding how parasite proteases mature from an inactive zymogen to an active enzyme is expected to inform new strategies for blocking their actions. Herein, we show that T. gondii cathepsin B protease (TgCPB) does not undergo self-maturation but instead requires the expression of a second papain-family cathepsin protease, TgCPL. Using recombinant enzymes we also show that TgCPL is capable of partially maturing TgCPB in vitro. Consistent with this interrelationship, antibodies with validated specificity detected TgCPB in the lysosome-like vacuolar compartment along with TgCPL. Our findings also establish that TgCPB does not localize to the rhoptries as previously reported. Accordingly, rhoptry morphology and rhoptry protein maturation are normal in TgCPB knock-out parasites. Finally, we show that although maturation of TgCPL is independent of TgCPB, it may involve an additional protease(s) in conjunction with self-maturation.