Fast compaction of alpha-lactalbumin during folding studied by stopped-flow X-ray scattering

To monitor the fast compaction process during protein folding, we have used a stopped-flow small-angle X-ray scattering technique combined with a two-dimensional charge-coupled device-based X-ray detector that makes it possible to improve the signal-to-noise ratio of data dramatically, and measured the kinetic refolding reaction of alpha-lactalbumin. The results clearly show that the radius of gyration and the overall shape of the kinetic folding intermediate of alpha-lactalbumin are the same as those of the molten globule state observed at equilibrium. Thus, the identity between the kinetic folding intermediate and the equilibrium molten globule state is firmly established. The present results also suggest that the folding intermediate is more hydrated than the native state and that the hydrated water molecules are dehydrated when specific side-chain packing is formed during the change from the molten globule to the native state.     

Role of tryptophan residues in the recognition of mutagenic oxidized nucleotides by human antimutator MTH1 protein

The human MTH1 antimutator protein hydrolyzes mutagenic oxidized nucleotides, and thus prevents their incorporation into DNA and any subsequent mutation. We have examined its great selectivity for oxidized nucleotides by analyzing the structure of the protein and its interaction with nucleotides, as reflected in the fluorescence of its tryptophan residues. The binding of nucleotides decreased the intensity of MTH1 protein fluorescence and red-shifted the emission peak, indicating that at least one tryptophan residue is close to the binding site. Oxidized nucleotides (2-OH-dATP and 8-oxo-dGTP) produced a larger decrease in fluorescence intensity than did unoxidized nucleotides, and MTH1 protein had a much higher binding affinity for oxidized nucleotides. Deconvolution of protein fluorescence by comparison of its quenching by positively (Cs(+)) and negatively (I(-)) charged ions indicated that the MTH1 tryptophan residues are in two different environments. One class of tryptophan residues is exposed to solvent but in a negatively charged environment; the other class is partially buried. While the binding of unoxidized nucleotides quenches the fluorescence of only class 1 tryptophan residue(s), the binding of oxidized nucleotides quenched that of class 2 tryptophan residue(s) as well. This suggests that selectivity is due to additional contact between the protein and the oxidized nucleotide. Mutation analysis indicated that the tryptophan residue at position 117, which is in a negative environment, is in contact with nucleotides. The negatively charged residues in the binding site probably correlate with the finding that nucleotide binding requires metal ions and depends upon their nature. Positively charged metal ions probably act by neutralizing the negatively charged nucleotide phosphate groups. (c) 2002 Elsevier Science Ltd.     

Developmental alteration of nerve injury induced glial cell line-derived neurotrophic factor (GDNF) receptor expression is crucial for the determination of injured motoneuron fate

Axotomy-induced neuronal death occurs in neonatal motoneurons, but not in adult rat. Here we demonstrated that during the course of postnatal development, nerve injury induced down-regulation of the glial cell line-derived neurotrophic factor (GDNF) receptor GFRalpha1 in axotomized hypoglossal motoneurons of rat are gradually converted to the adult up-regulation pattern of response. The compensatory expression of GFRalpha1 specifically in the injured motoneurons of neonates by adenovirus succeeded in rescuing the injured neurons without an application of growth factors. To the contrary, the nuclear antisense RNA for GFRalpha1 expression accelerates the axotomy-induced neuronal death in pups. These findings suggest that the receptor expression response after nerve injury is critical for the determination of injured motoneuron fate.     

Norepinephrine triggers Ca2+-dependent exocytosis of 5-hydroxytryptamine from rat pinealocytes in culture

5-hydroxytryptamine (5-HT) is a precursor and a putative modulator for melatonin synthesis in mammalian pinealocytes. 5-HT is present in organelles distinct from l-glutamate-containing synaptic-like microvesicles as well as in the cytoplasm of pinealocytes, and is secreted upon stimulation by norepinephrine (NE) to enhance serotonin N-acetyltransferase activity via the 5-HT2 receptor. However, the mechanism underlying the secretion of 5-HT from pinealocytes is unknown. In this study, we show that NE-evoked release of 5-HT is largely dependent on Ca2+ in rat pinealocytes in culture. Omission of Ca2+ from the medium and incubation of pineal cells with EGTA-tetraacetoxymethyl-ester inhibited by 59 and 97% the NE-evoked 5-HT release, respectively. Phenylephrine also triggered the Ca2+-dependent release of 5-HT, which was blocked by phentolamine, an alpha antagonist, but not by propranolol, a beta antagonist. Botulinum neurotoxin type E cleaved 25 kDa synaptosomal-associated protein and inhibited by 50% of the NE-evoked 5-HT release. Bafilomycin A1, an inhibitor of vacuolar H+-ATPase, and reserpine and tetrabenazine, inhibitors of vesicular monoamine transporter, all decreased the storage of vesicular 5-HT followed by inhibition of the NE-evoked 5-HT release. Agents that trigger L-glutamte exocytosis such as acetylcholine did not trigger any Ca2+-dependent 5-HT release. Vice versa neither NE nor phenylephrine caused synaptic-like microvesicle-mediated l-glutamate exocytosis. These results indicated that upon stimulation of a adrenoceptors pinealocytes secrete 5-HT through a Ca2+-dependent exocytotic mechanism, which is distinct from the exocytosis of synaptic-like microvesicles.     

The levels of mature glycosylated nicastrin are regulated and correlate with gamma-secretase processing of amyloid beta-precursor protein

Nicastrin, a type-I transmembrane glycoprotein, is a necessary component of the high molecular weight presenilin (PS) complexes that mediate intramembranous cleavage of beta-amyloid precursor protein (betaAPP) and Notch. Nicastrin undergoes trafficking-dependent glycosylation maturation, and PS1 interacts preferentially with these maturely glycosylated forms of nicastrin. We investigated the effects of differing levels of the immature and mature endoglycosidase-H-resistant forms of nicastrin on Abeta40- and Abeta42-peptide secretion in several cell lines stably expressing a mutant nicastrin (D336A/Y337A) that increases Abeta secretion. There was no correlation between Abeta secretion and the level of over-expression of the immature forms of nicastrin. The total level of mature nicastrin remained constant, but mutant nicastrin replaced endogenous mature nicastrin in varying degrees. Differences in the levels of mature mutant nicastrin positively correlated with Abeta secretion, but did not influence either betaAPP trafficking or processing by alpha- and beta-secretases. Proper trafficking and terminal maturation of nicastrin is therefore a necessary event for the regulated intramembranous proteolysis of betaAPP.     

Side-chain metabolism of propranolol: involvement of monoamine oxidase and mitochondrial aldehyde dehydrogenase in the metabolism of N-desisopropylpropranolol to naphthoxylactic acid in rat liver

We examined the metabolism of N-desisopropylpropranolol (NDP), which is generated from propranolol (PL) by side-chain N-desisopropylation, to naphthoxylactic acid (NLA) in rat liver. S(-)-NDP (S-NDP) and R(+)-NDP (R-NDP) were enantioselectively metabolized to NLA in isolated rat hepatocytes and in an enzyme reaction system of rat liver mitochondria with cofactor NAD+. Furthermore, the clearance profiles of NDP enantiomers were examined in an enzyme reaction system of rat liver mitochondria without NAD+. The amounts of S-NDP remaining in the incubation medium were similar to those of R-NDP, suggesting that monoamine oxidase (MAO) catalyzes the deamination of NDP to the aldehyde intermediate, but fails to deaminate enantioselectively S-NDP or R-NDP. Cyanamide, a potent inhibitor of aldehyde dehydrogenase (ALDH), markedly decreased the formation of NLA from racemic NDP in the enzyme reaction system of rat liver mitochondria with NAD+. When rat liver cytosol and microsomes were added to this enzyme reaction system, no significant alterations were observed in the amount of NLA generated from racemic NDP. We concluded that MAO deaminates NDP to an aldehyde intermediate, and that mitochondrial ALDH subsequently catalyzes the enantioselective metabolism of the aldehyde intermediate to NLA in rat liver.     

Proteases involved in long-term potentiation

Much attention has been paid to proteases involved in long-term potentiation (LTP). Calpains, Ca-dependent cysteine proteases, have first been demonstrated to be the mediator of LTP by the proteolytic cleavage of fodrin, which allows glutamate receptors located deep in the postsynaptic membrane to move to the surface. It is now generally considered that calpain activation is necessary for LTP formation in the cleavage of substrates such as protein kinase Czeta, NMDA receptors, and the glutamate receptor-interacting protein. Recent studies have shown that serine proteases such as tissue-type plasminogen activator (tPA), thrombin, and neuropsin are involved in LTP. tPA contributes to LTP by both receptor-mediated activation of cAMP-dependent protein kinase and the cleavage of NMDA receptors. Thrombin induces a proteolytic activation of PAR-1, resulting in activation of protein kinase C, which reduces the voltage-dependent Mg2+ blockade of NMDA receptor-channels. On the other hand, neuropsin may act as a regulatory molecule in LTP via its proteolytic degradation of extracellular matrix protein such as fibronectin. In addition to such neuronal proteases, proteases secreted from microglia such as tPA may also contribute to LTP. The enzymatic activity of each protease is strictly regulated by endogenous inhibitors and other factors in the brain. Once activated, proteases can irreversibly cleave peptide bonds. After cleavage, some substrates are inactivated and others are activated to gain new functions. Therefore, the issue to identify substrates for each protease is very important to understand the molecular basis of LTP.     

High glucuronidation activity of environmental estrogens in the carp (Cyprinus carpino) intestine

Many adverse effects on carp reproductive organs have been reported to be caused by exposure to environmental estrogens, such as nonylphenol and bisphenol A, which contaminate the aquatic environment. The glucuronidation activities of xenoestrogens (bisphenol A and diethylstilbestrol) and phytoestrogens (coumestrol, genistein and biochanin A), but not nonylphenol and octylphenol, were observed in microsomes prepared from carp organs. The highest levels of glucuronidation of environmental estrogens, for which the optimum temperature was 25-30 degrees C, were observed in the intestinal microsomes of 2-year-old carp. These activities in carp intestine increased developmentally, and the maximum levels corresponded to 5-10 % of that in rat liver microsomes. However, the glucuronidation of phytoestrogen by carp intestinal microsomes corresponded to that of rat liver microsomes. Only bisphenol A-glucuronide was excreted from the everted intestine, indicating that bisphenol A is metabolized in the carp intestine mainly as glucuronide.These results suggest that glucuronidation by carp intestine plays an important role for the detoxification of xenoestrogens and phytoestrogens, except for nonylphenol and octylphenol.     

Genotoxic activation of benzophenone and its two metabolites by human cytochrome P450s in SOS/umu assay

The genotoxic potential of benzophenone and its metabolically related compounds, benzhydrol and p-benzoylphenol, was investigated using human cytochrome P450 (P450) enzymes. Benzophenone and its two metabolites (0.1-1mM) showed a suppression of bacterial growth without any P450 system, but no induction of umu gene expression was observed in Salmonella typhimurium TA1535/pSK1002. Human liver microsomes induced the bacterial cytotoxicity of these compounds without any umu gene expression. On the other hand, with the addition of Escherichia coli membranes expressing recombinant human P450 2A6 and NADPH-cytochrome P450 reductase (NPR), benzophenone showed umu gene expression (64 umu units/min/nmol) P450 2A6). Moderate activation of benzophenone by P450 1A1/NPR membranes, 1A2/NPR membranes, or 1B1/NPR membranes was also observed. Activation of benzhydrol and p-benzoylphenol by the P450/NPR system was similar to that of benzophenone. These results suggest that benzophenone and its metabolically related benzhydrol and p-benzoylphenol can be bioactivated by P450 2A6 and P450 family 1 enzymes. Until now, benzophenone has been investigated mainly in terms of estrogenic activity and cytotoxicity, however, the genotoxic activation of benzophenone by human cytochrome P450s should be examined in terms of the risks to humans.     

Differential responses of Brassica napus and Petunia hybrida to leaf protoplast isolation stress

Changes in the response to abiotic stress during the isolation of leaf protoplasts were compared between a recalcitrant species of Brassica napus and regenerating species of Petunia hybrida . Initially, levels of soluble free putrescine (put), spermidine (spd) and spermine (spm) in leaves and protoplasts were determined. The sum of these three polyamines increased in petunia and B. napus leaf protoplasts by 1.6-fold and 1.1-fold, respectively. The soluble free fraction of spd and spm decreased in B. napus but not in petunia protoplasts. During the isolation of leaf protoplasts from B. napus , the ratio of soluble free put to the total PAs almost doubled, but that of spd and spm declined significantly. Petunia leaf protoplasts treated with cyclohexylamine (CHA), an inhibitor of spermidine synthase, accumulated ammonia and soluble putrescine, but lost the soluble spermidine. The soluble polyamine levels of CHA-treated petunia leaf protoplasts corresponded with those in B. napus . Leaves were subjected to abiotic stress during the isolation of protoplasts, namely wounding and osmotic stress which changed soluble free polyamine levels in B. napus and petunia, respectively. Both B. napus and petunia leaf protoplasts showed an increase in ammonia, but total free amino acid content and activation of proteases were only enhanced in B. napus leaf protoplasts. These results suggest that in B. napus wounding initiated senescence of leaf protoplasts during their isolation, leading to a constant production of ethylene early in the culture.