Poly(alpha-L-glutamic acid) in aqueous solution. Specific aggregation and hysteresis effects. II. Aggregation and precipitation. Chromatographic analysis

The aggregated form of poly(L -glutamic acid) can be isolated by gel permeation chromatography, whereby it was found that in a polydispersed sample the lower molecular weight fractions precipitate preferentially and the higher fractions tend instead to aggregate. In addition, aggregation has the effect of retarding or inhibiting precipitation.     

Specific aggregation of poly(α-L-glutamic acid) and hysteresis effects in aqueous solutions. I. Influence of temperature-dependent aggregation on the optical rotation of PGA

The instability of aqueous solutions of poly(α-L -glutamic acid) (PGA) at low pH is due to two distinguishable phenomena: precipitation, favored above 40°C., and aggregation, favored below 20°C. The aggregated form of PGA can be isolated by gel permeation chromatography. Both aggregation and precipitation increase with decreasing pH, i.e., with decreasing ionization of the side chain carboxyl groups. Temperature-induced aggregation and disaggregation give rise to a reproducible hysteresis loop which can be followed by optical rotation, light scattering, sedimentation, viscosity, and chromatography. Hysteresis has been observed with different PGA samples, and in several aqueous buffered or unbuffered solvents and organic-aqueous solvent mixtures and in the pH range 4.1–4.5. Aggregation manifests itself as an increase in negative optical rotation in the visible and ultraviolet spectral range. The specific relation at 233 mμ is sensitive to aggregation and also reflects the hysteresis. Measurements of optical rotatory dispersion indicate that a₀ reflects the hysteresis but b₀ does not, the latter revealing only reversible changes with aggregation and disaggregation. The helix-coil equilibrium is apparently unperturbed by aggregation, as is the thermal stability of the helix structure. For fully aggregated PGA it is estimated that a₀ increases by about 300 degrees, which suggests that a₀ may be a sensitive parameter to measure aggregation in other systems. The rate of aggregation increases with decreasing temperature. The disaggregation, upon heating, is more rapid. However, kinetics measurements have not yet been done. The temperature M at which all aggregates are disrupted increases with decreasing pH, but is independent of total PGA concentration, at constant pH. No molecular weight dependence of M was detected in the range 20–100 × 10³. The shape and size of the hysteresis loop depends upon pH and molecular weight, which is interpreted as a dependence on the extent of aggregation. One branch of the loop, representing the helix–coil transition of isolated molecules, is reversible, while the others, representing the formation and disruption of the aggregates, are not. The system exhibits both ascending and descending scanning curves, which are typical of a true hysteresis.     

Mathematical Analysis of Haemopoietic Grafted Cell Renewal and Migration Through the Allogeneic Or Syngeneic Mouse Host

Abstract. A mathematical analysis of results from kinetic studies of 125-iododeoxyuri-dine uptake and loss in almost all the lymphoid and non-lymphoid organs of mice is described. Applied to data gathered from a graft- versus -host reaction experiment, this analysis affords quantitative precision on the differential effects of organ alloantigens on the proliferating grafted cells. It is shown that, depending on the organ and the post-graft period, cell growth can be ascribed to alloantigen-driven cell renewal or to alloantigen-driven trapping or sequestration. Possible applications of the present approach in graft rejection monitoring are discussed.     

Ion channel stabilization of synthetic alamethicin analogs by rings of inter-helix H-bonds.

Rings of inter-helix H-bonds due to Gln at position 7, a highly conserved residue in all pore-forming peptaibols, have been suggested to play an important role in the stabilization of alamethicin channels. In an attempt to test this hypothesis, experimental studies have been undertaken on four synthetic alamethicin non-Aib analogs (Alm-dUL) in which the Gln at position 7 (Q7) is substituted by Ala, Asn, or Ser (Q7A, Q7N, or Q7S). Voltage-dependent pore formation by these analogs in planar lipid bilayers is compared at the macroscopic and single-channel conductance levels. As anticipated, the Q7A substitution abolished all channel-forming activity. The voltage dependence of macroscopic current-voltage curves was conserved with the Q7N substitution but reduced in the Q7S analog. Normalized single-channel conductance ratios between substates follow the same pattern, with the Q7S analog yielding the highest unit conductances. Channel lifetimes were the most significantly modulated parameter with markedly faster kinetics when Gln or Asn was replaced by Ser. The effect of the Q7S substitution on channel lifetimes may be explained through a reduced stabilization of bundles by inter-helix H-bonds.     

IL-10 signaling is essential for 1,25-dihydroxyvitamin D3-mediated inhibition of experimental autoimmune encephalomyelitis

Multiple sclerosis (MS) results from an aberrant, neuroantigen-specific, T cell-mediated autoimmune response. Because MS prevalence and severity decrease sharply with increasing sunlight exposure, and sunlight supports vitamin D(3) synthesis, we proposed that vitamin D(3) and 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)) may protect against MS. In support of this hypothesis, 1,25-(OH)(2)D(3) strongly inhibited experimental autoimmune encephalomyelitis (EAE). This inhibition required lymphocytes other than the encephalitogenic T cells. In this study, we tested the hypothesis that 1,25-(OH)(2)D(3) might inhibit EAE through the action of IL-10-producing regulatory lymphocytes. We report that vitamin D(3) and 1,25-(OH)(2)D(3) strongly inhibited myelin oligodendrocyte peptide (MOG(35-55))-induced EAE in C57BL/6 mice, but completely failed to inhibit EAE in mice with a disrupted IL-10 or IL-10R gene. Thus, a functional IL-10-IL-10R pathway was essential for 1,25-(OH)(2)D(3) to inhibit EAE. The 1,25-(OH)(2)D(3) also failed to inhibit EAE in reciprocal, mixed bone marrow chimeras constructed by transferring IL-10-deficient bone marrow into irradiated wild-type mice and vice versa. Thus, 1,25-(OH)(2)D(3) may be enhancing an anti-inflammatory loop involving hemopoietic cell-produced IL-10 acting on brain parenchymal cells and vice versa. If this interpretation is correct, and humans have a similar bidirectional IL-10-dependent loop, then an IL-10-IL-10R pathway defect could abrogate the anti-inflammatory and neuro-protective functions of sunlight and vitamin D(3). In this way, a genetic IL-10-IL-10R pathway defect could interact with an environmental risk factor, vitamin D(3) insufficiency, to increase MS risk and severity.     

Weight–length relationship of 63 demersal fishes on the shallow coast of Paraná, Brazil

This work presents the weight–length relationship of 63 species of fish belonging to 24 families. Data were collected monthly along the Paraná state coast (Brazil) from August 2004 to July 2005 on five transects between 6 and 15 m. Several of these species had no previously published weight–length relationships.     

Examination of the Cytotoxic and Embryotoxic Potential and Underlying Mechanisms of Next-Generation Synthetic Trioxolane and Tetraoxane Antimalarials

Semisynthetic artemisinin-based therapies are the first-line treatment for P. falciparum malaria, but next-generation synthetic drug candidates are urgently required to improve availability and respond to the emergence of artemisinin-resistant parasites. Artemisinins are embryotoxic in animal models and induce apoptosis in sensitive mammalian cells. Understanding the cytotoxic propensities of antimalarial drug candidates is crucial to their successful development and utilization. Here, we demonstrate that, similarly to the model artemisinin artesunate (ARS), a synthetic tetraoxane drug candidate (RKA182) and a trioxolane equivalent (FBEG100) induce embryotoxicity and depletion of primitive erythroblasts in a rodent model. We also show that RKA182, FBEG100 and ARS are cytotoxic toward a panel of established and primary human cell lines, with caspase-dependent apoptosis and caspase-independent necrosis underlying the induction of cell death. Although the toxic effects of RKA182 and FBEG100 proceed more rapidly and are relatively less cell-selective than that of ARS, all three compounds are shown to be dependent upon heme, iron and oxidative stress for their ability to induce cell death. However, in contrast to previously studied artemisinins, the toxicity of RKA182 and FBEG100 is shown to be independent of general chemical decomposition. Although tetraoxanes and trioxolanes have shown promise as next-generation antimalarials, the data described here indicate that adverse effects associated with artemisinins, including embryotoxicity, cannot be ruled out with these novel compounds, and a full understanding of their toxicological actions will be central to the continuing design and development of safe and effective drug candidates which could prove important in the fight against malaria.