Feedback control of the protein kinase TAK1 by SAPK2a/p38alpha

TAB1, a subunit of the kinase TAK1, was phosphorylated by SAPK2a/p38alpha at Ser423, Thr431 and Ser438 in vitro. TAB1 became phosphorylated at all three sites when cells were exposed to cellular stresses, or stimulated with tumour necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1) or lipopolysaccharide (LPS). The phosphorylation of Ser423 and Thr431 was prevented if cells were pre-incubated with SB 203580, while the phosphorylation of Ser438 was partially inhibited by PD 184352. Ser423 is the first residue phosphorylated by SAPK2a/p38alpha that is not followed by proline. The activation of TAK1 was enhanced by SB 203580 in LPS-stimulated macrophages, and by proinflammatory cytokines or osmotic shock in epithelial KB cells or embryonic fibroblasts. The activation of TAK1 by TNF-alpha, IL-1 or osmotic shock was also enhanced in embryonic fibroblasts from SAPK2a/p38alpha-deficient mice, while incubation of these cells with SB 203580 had no effect. Our results suggest that TAB1 participates in a SAPK2a/p38alpha-mediated feedback control of TAK1, which not only limits the activation of SAPK2a/p38alpha but synchronizes its activity with other signalling pathways that lie downstream of TAK1 (JNK and IKK).     

Lipid-alginate interactions render changes in phospholipid bilayer permeability

Lipid vesicles, e.g. liposomes, generally release their contents in a continuous manner. However, when these vesicles are entrapped in Ca-alginate and coated with poly(L-lysine), they release their contents in an unusual fashion, in 'bursts'. Molecular-level studies indicated that lipid-alginate interactions are responsible for changes in the barrier properties of lipid vesicles. Differential scanning calorimetry revealed that exposure of liposomes to alginate resulted in a 4-fold reduction in the phase transition enthalpy, with no change in the melting temperature. Size-exclusion chromatography of liposomes-in-alginate gave an additional liposomal peak with a smaller elution volume. These studies suggested that alginate is inserted into the lipid bilayer of vesicles. Lipid-alginate interactions were highly dependent on phospholipid head group charge and the phase transition temperature of the phospholipid. Based on these interactions, a mechanism to explain the 'burst' from these entrapped liposomes is suggested.     

Kinetics of the interaction of N-(phosphonacetyl)-L-aspartate with the catalytic subunit of aspartate transcarbamoylase. A slow conformational change subsequent to binding

The binding of the bisubstrate ligand N-(phosphonacetyl)-L-aspartate (PALA) to the active sites of both the free catalytic subunit of aspartate transcarbamoylase and the intact holoenzyme causes conformational changes which have been studied extensively. However, no kinetic information has been available about the sequence of events occurring during the formation or dissociation of the complexes. Stopped flow kinetics, 31P saturation transfer NMR spectroscopy, and presteady-state kinetics were used to monitor the interaction of PALA with the catalytic subunit (or a derivative containing nitrotyrosyl chromophores which served as spectral probes). The various experimental approaches lead to a mechanism that includes a rapid binding of PALA with an "on" rate of about 10(8)M-1s-1 and an "off" rate of 28 s-1, followed by a much slower isomerization of the complex with a forward rate constant of 0.18 s-1. Analysis of the presteady-state bursts of enzyme activity when the protein is added to a mixture of substrates and PALA and of the lag in activity when the PALA complex with catalytic subunit is added to substrates yielded a rate constant for the reverse isomerization of 0.018s-1. Thus, the conformational change subsequent to PALA binding leads to a 10-fold increase in the equilibrium constant for complex formation. Stopped flow kinetic measurements of the spectral change resulting from mixing the complex of PALA and nitrated protein with native enzyme showed a slow process with a t1/2 of about 11 s, whereas 31P saturation transfer NMR experiments yielded at t1/2 of about 260 ms for the dissociation of PALA from the complex. This apparent disparity is understood in terms of the two-step binding scheme where rapid dissociation of the initial ligand X enzyme complex is measured by the NMR technique and the slow isomerization of the complex is responsible for the bulk of the stopped flow signal.     

Formation and function of the polar body contractile ring in Spisula

Initial studies suggested that spatial organization of the putative polar body contractile ring was determined by the peripheral aster in Spisula [Biol. Bull. 205 (2003) 192]. Here we report detailed supporting observations, including testing of aster and ring function with inhibitors. The metaphase peripheral aster was confirmed to spread cortically in an umbrella-like pattern, with microtubule-poor center. The aster disassembled during anaphase, leaving the spindle docked at the F-actin-poor center of a newly generated cortical F-actin ring that closely approximated the aster in location, measured diameter range, and pattern. Cytochalasin D and latrunculin-B permitted all events except ring and polar body formation. Nocodazole disassembly or taxol stabilization of the peripheral aster produced poorly defined rings or bulging anaphase asters within the ring center, respectively, inhibiting polar body formation. Polar body extrusion occurred at the ring center, the diameter of which diminished. Ring contractility-previously assumed-was verified using blebbistatin, a myosin-II ATPase inhibitor that permitted ring assembly but blocked polar body extrusion. The data support the hypothesis that peripheral aster spreading, perhaps dynein-driven, is causally related to polar body contractile ring formation, with anaphase entry and aster disassembly also required for polar body biogenesis. Previously reported astral spreading during embryonic micromere formation suggests that related mechanisms are involved in asymmetric somatic cytokinesis.     

Antigenic analysis of a major neutralization site of herpes simplex virus glycoprotein D, using deletion mutants and monoclonal antibody-resistant mutants.

Herpes simplex virus glycoprotein D is a component of the virion envelope and appears to be involved in attachment, penetration, and cell fusion. Monoclonal antibodies against this protein can be arranged in groups on the basis of a number of biological and biochemical properties. Group I antibodies are type common, have high complement-independent neutralization titers, and recognize discontinuous (conformational) epitopes; they are currently being used in several laboratories to study the functions of glycoprotein D. We have used a panel of neutralization-resistant mutants to examine the relationships between these antibodies in detail. We found that they can be divided into two subgroups, Ia and Ib, such that mutations selected with Ia antibodies have little or no effect on binding and neutralization by Ib antibodies and vice versa. In addition, Ia antibodies are able to bind deletion and truncation mutants of glycoprotein D that Ib antibodies do not recognize, suggesting that their epitopes are physically distinct. However, with one exception, Ia and Ib antibodies block each other strongly in binding assays with purified glycoprotein D, whereas antibodies from other groups have no effect. We have therefore defined the sum of the Ia and Ib epitopes as antigenic site 1.     

Independent activation of subfornical organ and hypothalamo-neurohypophysial system during administration of angiotensin II

The autoradiographic deoxyglucose method was employed to investigate: 1) whether the increased glucose utilization in the subfornical organ (SFO) during administration of angiotensin II (AII) depends on the neural inputs to the SFO; and 2) to investigate whether the activation of the hypothalamo-neurohypophysial system during administration of AII depends on inputs from the SFO. The ventral stalk of the SFO, which contains the majority of efferent and afferent projections of this circumventricular structure, was interrupted with knife cuts three days before the deoxyglucose experiments. Intravenous infusion of AII (2.5 micrograms/min) for 45 min increased glucose utilization in the SFO and neural lobe in the lesioned animals to the same extent as in the sham-operated animals. Drinking, however, was significantly reduced in lesioned animals. These experiments disclose independent parallel mechanisms responsible for activation of the SFO and the hypothalamo-neurohypophysial system by AII.     

Deprenyl Protects Dopamine Neurons from the Neurotoxic Effect of 1-Methyl-4-Phenylpyridinium Ion

1-Methyl-4-phenylpyridinium ion (MPP+) is the product of the metabolic oxidation of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) by monoamine oxidase (MAO). MPP+ is toxic to 3,4-dihydroxyphenylethylamine (dopamine, DA) neurons in explant cultures of rat embryonic midbrain. Addition of 2.5 microM MPP+ to the feeding medium for 6 days results in significant reduction of the DA levels in the cultures (to 19% of control) as well as in the uptake of [3H]DA (to 32% of control). When the cultures are treated with the MAO inhibitor deprenyl (10 microM) 24 h prior to and during exposure to MPP+, the DA neurons are protected from the toxicity of the drug. In the combined deprenyl plus MPP+ treatment, the levels of DA in the cultures remain at the control range and the [3H]DA uptake is reduced to only 73% of control. These results indicate that MAO is involved in the toxicity of MPP+ on DA neurons.