Physical and biological parameters that determine the fate of p-chlorophenol in laboratory test systems

Shake-flask and microcosm studies were conducted to determine the fate of para-chlorophenol (p-CP) in water and sediment systems and the role of sediment and nonsediment surfaces in the biodegradation process. Biodegradation of p-CP in estuarine water samples in shake flasks was slow over incubation periods of 300 h. The addition of detrital sediment resulted in immediate and rapid degradation evidenced by the production of 14CO2 from [14C]p-CP. The addition of sterile sediment, glass beads, or sand resulted in approximately four to six times more CO2 evolution than observed in the water alone. Densities of p-CP-degrading bacteria associated with the detrital sediment were 100 times greater than those enumerated in water. Bacteria in the water and associated with the sediment after preexposure of both water and sediment of p-CP demonstrated enhanced biodegradation. In some microcosms, p-CP was degraded completely in the top 1.0 cm of intact sediment beds. Sediment reworking activities by benthic invertebrates from one site were sufficient to mix p-CP deep into the sediment bed faster than biodegradation or molecular diffusion. p-CP was persistent at lower depths of the sediment, possibly a result of reduced oxygen conditions preventing aerobic biodegradation.     

The generation and subsequent fate of glutathionyl radicals in biological systems

Horseradish peroxidase-catalyzed oxidation of p-phenetidine in the presence of either glutathione (GSH), cysteine, or N-acetylcysteine led to the production of the appropriate thioyl radical which could be observed using EPR spectroscopy in conjunction with the spin trap 5,5-dimethyl-1-pyrroline-N-oxide. This confirms earlier work using acetaminophen (Ross, D., Albano, E., Nilsson, U., and Moldéus, P. (1984) Biochem. Biophys. Res. Commun. 125, 109-115). The further reactions of glutathionyl radicals (GS.), generated during horseradish peroxidase-catalyzed oxidation of p-phenetidine and acetaminophen in the presence of GSH, were investigated by following kinetics of oxygen uptake and oxidized glutathione (GSSG) formation. Oxygen uptake and GSSG generation were dependent on the concentration of GSH but above that which was required for maximal interaction with the primary amine or phenoxy radical generated during peroxidatic oxidation of p-phenetidine or acetaminophen, suggesting that a secondary GSH-dependent process was responsible for oxygen uptake and GSSG production. GSSG was the only product of thiol oxidation detected during peroxidatic oxidation of p-phenetidine or acetaminophen in the presence of GSH, but under nitrogen saturation conditions its production was reduced to 8 and 33% of the corresponding amounts obtained under aerobic conditions in the cases of p-phenetidine and acetaminophen, respectively. Nitrogen saturation conditions did not affect horseradish peroxidase-catalyzed metabolism. This shows that the main route of GSSG generation in such reactions is not by dimerization of GS. but via mechanism(s) involving oxygen consumption such as via GSSG-. or via GSOOH.     

Physical problems with the vitrification of large biological systems

Vitrification is an attractive potential pathway to the successful cryopreservation of mature mammalian organs, but modern cryobiological research on vitrification to date has been devoted mostly to experiments with solutions and with biological systems ranging in diameter from about 6 through about 100 microns. The present paper focuses on concerns which are particularly relevant to large biological systems, i.e., those systems ranging in size from approximately 10 ml to approximately 1.5 liters. New qualitative data are provided on the effect of sample size on the probability of nucleation and the ultimate size of the resulting ice crystals as well as on the probability of fracture at or below Tg. Nucleation, crystal growth, and fracture depend on cooling velocity and the magnitude of thermal gradients in the sample, which in turn depend on sample size, geometry, and cooling technique (environmental thermal history and thermal uniformity). Quantitative data on thermal gradients, cooling rates, and fracture temperatures are provided as a function of sample size. The main conclusions are as follows. First, cooling rate (from about 0.2 to about 2.5 degrees C/min) has a profound influence on the temperature-dependent processes of nucleation and crystal growth in 47-50% (w/w) solutions of propylene glycol. Second, fracturing depends strongly on cooling rate and thermal uniformity and can be postponed to about 25 degrees C below Tg for a 482-ml sample if cooling is slow and uniform. Third, the presence of a carrier solution reduces the concentration of cryoprotectant needed for vitrification (CV). However, the CV of samples larger than about 10 ml is significantly higher than the CV of smaller samples whether a carrier solution is present or not.     

A quantitative test for superoxide radicals produced in biological systems.

The preparation and properties of a partially succinoylated cytochrome c, suited for the detection of superoxide anion radicals in liver microsomes, is reported. By succinoylation of 45% of the primary amino groups of horse heart cytochrome c the activity towards solubilized NADPH--cytochrome P-450 reductase was diminished by 99% compared with native cytochrome c. The capacities of cytochrome b5 and cytochrome c oxidase to reduce the succinoylated ferricytochrome c and oxidize succinoylated ferrocytochrome c respectively were decreased to a similar extent. However, the bimolecular rate constant for the reduction of the partially succinoylated ferricytochrome c by O2-. was estimated to be one-tenth of the value for the reaction of O2-. with native ferricytochrome c a pH 7.7. On this basis the quantification of O2-. generated by NADPH-supplemented liver microsomes became possible. The initial rates of succinoylated ferricytochrome c reduction determined at various finite concentrations of the cytochrome c derivative can be extrapolated to obtain true rates of O2-. generation in a homogeneous system. The problems encountered in the quantitative determination of O2-. produced in biological membranes, e.g. microsomes, are discussed.     

A simplified procedure to determine the optimal rate of freezing biological systems

The effect of several cell-level parameters on the predicted optimal cooling rate B(opt) of an arbitrary biological system has been studied using a well-defined water transport model. An extensive investigation of the water transport model revealed three key cell level parameters: reference permeability of the membrane to water L(pg), apparent activation energy E(Lp), and the ratio of the available surface area for water transport to the initial volume of intracellular water (SA/WV). We defined B(opt) as the "highest" cooling rate at which a predefined percent of the initial water volume is trapped inside the cell (values ranging from 5% to 80%) at a predefined end temperature (values ranging from -5 degrees C to -40 degrees C). Irrespective of the choice of the percent of initial water volume trapped and the end temperature, an exact and linear relationship exists between L(pg), SA/WV, and B(opt0. However, a nonlinear and inverse relationship is found between E(Lp) and B(opt). Remarkably, for a variety of biological systems a comparison of the published experimentally determined values of B(opt) agreed quite closely with numerically predicted B(opt) values when the model assumed 5% of initial water is trapped inside the cell at a temperature of -15 degrees C. This close agreement between the experimental and model predicted optimal cooling rates is used to develop a generic optimal cooling rate chart and a generic optimal cooling rate equation that greatly simplifies the prediction of the optimal rate of freezing of biological systems.     

SIT and TRIM determine T cell fate in the thymus

Thymic selection is a tightly regulated developmental process essential for establishing central tolerance. The intensity of TCR-mediated signaling is a key factor for determining cell fate in the thymus. It is widely accepted that low-intensity signals result in positive selection, whereas high-intensity signals induce negative selection. Transmembrane adaptor proteins have been demonstrated to be important regulators of T cell activation. However, little is known about their role during T cell development. Herein, we show that SIT (SHP2 Src homology domain containing tyrosine phosphatase 2-interacting transmembrane adaptor protein) and TRIM (TCR-interacting molecule), two structurally related transmembrane adaptors, cooperatively regulate TCR signaling potential, thereby influencing the outcome of thymic selection. Indeed, loss of both SIT and TRIM resulted in the up-regulation of CD5, CD69, and TCRbeta, strong MAPK activation, and, consequently, enhanced positive selection. Moreover, by crossing SIT/TRIM double-deficient mice onto transgenic mice bearing TCRs with different avidity/affinity, we found profound alterations in T cell development. Indeed, in female HY TCR transgenic mice, positive selection was completely converted into negative selection resulting in small thymi devoided of double-positive thymocytes. More strikingly, in a nonselecting background, SIT/TRIM double-deficient single-positive T cells developed, were functional, and populated the periphery. In summary, we demonstrated that SIT and TRIM regulate cell fate of developing thymocytes, thus identifying them as essential regulators of central tolerance.     

Transplantation of somatic nuclei into oocyte cytoplasm reveals that the chromosome properties determine the chromosome separation fate in rabbit

G2/M somatic nuclei were introduced into enucleated meiotically competent oocytes and subsequently cultured in TCM199 plus 10% fetal calf serum (FCS). Pseudo-first polar bodies could be extruded, but the chromosomes failed to arrange normally. Kinetochores were traced with immunofluorescent microscopy using autoimmune sera from patients with CREST (Calcinosis, Raynaud's phenomenon, Esophageal dysmotility, Sclerodactyly, Telangiectasia) scleroderma. In vitro matured oocytes arrested at second meiotic metaphase and kinetochores were detectable as paired structures aligned at the spindle equator. At meiotic anaphase, present or past the kinetochores separated and remained aligned at the distal sides of the chromosomes until telophase, when their alignment perpendicular to the spindle axis was lost. Kinetochores failed to arrange normally after transferring somatic nuclei into oocytes. Our results suggest that somatic cell nuclei are unable to proceed normally through meiosis when introduced into oocyte meiotic cytoplasm.     

Mechanisms that determine the transmembrane disposition of proteins.

The final orientation that a protein assumes in the membrane of the endoplasmic reticulum is determined by a few types of signal sequences and their respective interactions with the membrane insertion complex. Membrane insertion occurs via a series of discrete steps, some of which are regulated by GTP- and ATP-binding proteins. Analysis of the protein components in proximity to nascent secretory and membrane proteins has revealed novel proteins in the endoplasmic reticulum that may form part of the membrane insertion complex.     

Solid-state NMR investigations of interaction contributions that determine the alignment of helical polypeptides in biological membranes

Helical peptides reconstituted into oriented phospholipid bilayers were studied by proton-decoupled 15N solid-state NMR spectroscopy. Whereas hydrophobic channel peptides, such as the N-terminal region of Vpu of HIV-1, adopt transmembrane orientations, amphipathic peptide antibiotics are oriented parallel to the bilayer surface. The interaction contributions that determine the alignment of helical peptides in lipid membranes were analysed using model sequences, and peptides that change their topology in a pH-dependent manner have been designed. The energy contributions of histidines, lysines, leucines and alanines as well as the alignment of peptides and phospholipids under conditions of hydrophobic mismatch have been investigated in considerable detail.     

Molecular markers to determine the ecological fate of Bacillus thuringiensis ssp. kurstaki

A set of DNA markers was developed that successfully identifies Bacillus thuringiensis ssp. kurstaki (Btk) when screened against other Bacillus species and subspecies. These subspecies-specific primer sets allowed detection and characterization of Btk within an environmental background that contained many Bacillus species. Because Btk is used as an active ingredient in many commercial formulations, yet is not naturally widely distributed in North America or Europe, these markers will prove useful in investigations on the environmental persistence and ecological fate of Btk.     

Physical properties of erythrocyte ghosts that determine susceptibility to secretory phospholipase A2

Artificial membranes may be resistant or susceptible to catalytic attack by secretory phospholipase A(2) (sPLA(2)) depending on the physical properties of the membrane. Living cells are normally resistant but become susceptible during trauma, apoptosis, and/or a significant elevation of intracellular calcium. Intact erythrocytes and ghosts were studied to determine whether the principles learned from artificial systems apply to biological membranes. Membrane properties such as phospholipid and/or protein composition, morphology, and microscopic characteristics (e.g. fluidity) were manipulated by preparing ghosts under different experimental conditions such as in the presence or absence of divalent cations with or without ATP. The properties of each membrane preparation were assessed by biochemical and physical means (fluorescence spectroscopy and electron and two-photon microscopy using the membrane probes bis-pyrene and laurdan) and compared with sPLA(2) activity. The properties that appeared most relevant were the degree of phosphatidylserine exposure on the outer face of the membrane and changes to the membrane physical state detected by bis-pyrene and laurdan. Specifically, vulnerability to hydrolysis by sPLA(2) was associated with an increase in bilayer order apparently reflective of expansion of membrane regions of diminished fluidity. These results argue that the general principles identified from studies with artificial membranes apply to biological systems.     

The fate of phosphate under anoxic conditions in biological nutrient removal activated sludge systems

The nitrogen removal potential of phosphate accumulating organisms under anoxic conditions has been evaluated using a laboratory scale sequencing batch reactor fed with synthetic wastewater and operated in a sequence of anaerobic, anoxic and aerobic periods. The phosphate uptake rate under anoxic conditions was lower than that under aerobic conditions. However, in the presence of an external substrate such as glucose and acetate, the fate of phosphate was dependent on the substrate type; phosphate release occurred in the presence of nitrate as long as acetate was present and glucose did not cause any phosphate release. The nitrate uptake rate was also much lower with glucose than acetate. The results implied that poly-hydroxyalkanoates could be oxidized by nitrate and phosphate uptake during the anoxic phase should be introduced into process modeling. © Rapid Science Ltd. 1998     

Regulatory modules that generate biphasic signal response in biological systems

Biochemical networks might be composed of modules. It is still not clear how biochemical modules can be defined and characterised. Here we propose a functional approach to module definition, considering different classes of biphasic regulation modules, which effect optimal cell response to intermediate signal strength. Each regulation class might possess unique properties that make it especially suitable for particular biological functions.     

A thermodynamic optimization analysis of a possible relation between the parameters that determine the energetics of muscle contraction in steady state.

Given the phenomenological relations for muscle's steady-state contraction and proper definitions of power p and efficiency eta, the behavior of these quantities is analysed in terms of the parameters that determine the energetics of the muscle, here denoted by s(o)and alpha. s(o)is proportional to the so-called maintenance heat, while alpha is the parameter that determines the curvature of the Hill's force-velocity curve. The dependence of the muscle's power and efficiency, averaged over the whole range of force the muscle can exert, on the parameters s(o)and alpha is studied. The average power p(avg)is a function only of alpha, and is a growing function that approaches 1/6 asymptotically as alpha goes to infinity. The average efficiency eta(avg)is a function of both alpha and s(o). With the value of s(o)fixed, the graph of the function eta(avg)(s(o), alpha) is a convex curve with a single maximum. The value and the position of this maximum point both depend on s(o). In the limit alpha-->0, s(o)-->0, eta(avg)tends to 1. The points (s(o), alpha(m)(s(o))), with alpha(m)(s(o)) the value of alpha that maximizes eta(avg)for a given s(o), are fitted by the curve alpha=s(o 1/2). This relation was experimentally found by A.V. Hill in his early studies of muscle energetics. Other experimental data are found to qualitatively satisfy the same relation. Although some dynamical microscopic models for muscle contraction, based upon Huxley's cross-bridge model, show that the same kinetic parameters control both the maintenance heat (s(o)) and the muscle's power output (alpha), we suggest that the exact relation between them has been reached due to the evolutive stresses that made individuals with equally powerful and more efficient muscles more suitable to reproduce.