We have shown previously that glycogen synthase kinase-3␤ (GSK-3␤), cyclin-dependent kinase 5, and c-Jun NH2-terminal kinase become overactivated and hyperphosphorylate in heat-shocked female rats. This hyperphosphorylation of is estrogen-independent, prevented by androgens, and similar to Alzheimer's disease. In this study, ovariectomized (OVX) Sprague-Dawley rats (n ‫؍‬ 75) received daily injections of 10 g of 17␤-estradiol benzoate (EB), or 250 g of testosterone propionate (TP), or both EB and TP, or sesame oil (SO) vehicle for 4 -6 weeks. In kinase assays of forebrain homogenates, overactivation of GSK-3␤ at 0 -6 h after heat shock toward human recombinant , bovine , and phosphoglycogen synthase peptide 2 was prevented in OVX ؉ TP and OVX ؉ (EB ؉ TP) but not in sham-OVX ؉ SO, OVX ؉ SO, and OVX ؉ EB. Abs against inactive (pSer 9 ) and activity-enhanced (pTyr 216 ) GSK-3␤ showed marked increase of pSer 9 -and decrease of pTyr 216 -GSK-3␤ in both OVX ؉ TP and OVX ؉ (EB ؉ TP) but not in sham-OVX ؉ SO, OVX ؉ SO, and OVX ؉ EB. EB enhanced the overactivation of cyclin-dependent kinase 5. The activity of c-Jun NH2-terminal kinase was gonadal hormone-independent. The serum concentrations of testosterone and 17␤-estradiol were 2.53 ng͞ml and 201 pg͞ml in OVX ؉ TP and OVX ؉ EB, respectively. These findings demonstrate that testosterone prevents the hyperphosphorylation of by inhibiting the heat shock-induced overactivation of GSK-3␤ and suggest that androgens given to aging men or, in combination with estrogens, to postmenopausal women could prevent or delay Alzheimer's disease.A bout two times more women than men have Alzheimer's disease (AD) (1), partly because women with AD live longer. However, recent studies showed that women carry an innate higher risk for AD (2). The precipitous decline and loss of neuroprotective effects of estrogens in postmenopausal women-in contrast to the gradual decline of androgens in aging men-are offered as an explanation. However, recent studies showed no beneficial effects of estrogens on mild-to-moderate AD (3). On the other hand, a possible advantageous role of androgens in the prevention and͞or treatment of AD has not been tried yet despite their neuroprotective effects (4), the century-old suggestion that they may rejuvenate aged men (5), lower serum testosterone concentration in men with AD (6), the increasing evidence that stressful stimuli play a role in the etiopathogenesis of AD (7,8), and inhibition of stress response by androgens (9).The cause of AD is not known, but it seems to be a syndrome resulting from an interplay among a genetic predisposition, environmental stress factors, and the aging process. The histologic hallmarks of AD are the senile plaques made of A␤ amyloid, dystrophic neurites, and reactive glial cells, and the neurofibrillary tangles composed of bundles of abnormal filaments, the so-called paired-helical filaments, the major component of which is hyperphosphorylated (10-12). However, the earliest manifestation of hyperphosphorylated is a granular form in...

We have previously shown, by using the phosphate-dependent anti-antibodies Tau-1 and PHF-1, that heat shock induces rapid dephosphorylation of followed by hyperphosphorylation in female rats. In this study, we analyzed in forebrain homogenates from female Sprague-Dawley rats the activities of extracellular signal regulated kinase 1͞2 (ERK1͞2), c-Jun NH2-terminal kinase (JNK), glycogen synthase kinase-3␤ (GSK-3␤), cyclin-dependent kinase 5 (Cdk5), cAMP-dependent protein kinase A (PKA), and Ca 2؉ ͞calmodulin-dependent protein kinase II (CaMKII) at 0 (n ‫؍‬ 5), 3 (n ‫؍‬ 4), 6 (n ‫؍‬ 5), and 12 (n ‫؍‬ 5) h after heat shock and in non-heat-shocked controls (n ‫؍‬ 5). Immunoprecipitation kinase assays at 0 h showed suppression of the activities of all kinases except of GSK-3␤, which showed increased activity. At 3-6 h, the activities of ERK1͞2, JNK, Cdk5, and GSK-3␤ toward selective substrates were increased; however, only JNK, Cdk5, and GSK-3␤ but not ERK1͞2 were overactivated toward purified bovine . At 3-6 h, kinase assays specific for PKA and CaMKII showed no increased activity toward either or selective substrates. All of eight anti-antibodies tested showed dephosphorylation at 0 h and hyperphosphorylation at 3-6 h, except for 12E8, which showed hyperphosphorylation also at 0 h. Immunoblot analysis using activity-dependent antibodies against ERK1͞2, JNK, and GSK-3␤ confirmed the above data. Increased activation and inhibition of kinases after heat shock were statistically significant in comparison with controls. Because is hyperphosphorylated in Alzheimer disease these findings suggest that JNK, GSK-3␤, and Cdk5 may play a role in its pathogenesis.

In the present investigation, in vitro phosphorylation of CNS proteins of the silkworm Bombyx mori during the postembryonic development have been studied. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and autoradiography of phosphorylated proteins revealed the presence of major phosphoproteins of 59/60 kDa. Based on molecular mass, calcium/calmodulin‐dependent autophosphorylation, substrate specificity, KN‐62 inhibition, apparent Km for ATP and syntide‐2, these proteins were identified as calcium/calmodulin‐dependent protein kinase II (CaM kinase II). Anti‐rat CaM kinase II monoclonal antibody showed immunoreactivity with Bombyx CaM kinase II isoforms. This kinase showed a high degree of autophosphorylation in neural tissue. During postembryonic development of Bombyx, two distinct peaks of enzyme activity could be noticed, one at the late‐larval and another at the late‐pupal stage, which were associated with an increase in amount of the enzyme. These results suggested that the expression of CaM kinase II in the CNS of Bombyx was developmentally regulated.

Protein fractions from the whole body homogenates were separated on SDS-PAGE and the muscle actin band was identified by western blotting, using commercial antibody raised against chick back muscle actin (Sigma Chemical Co., USA). The antibody was found to be specific for muscle actin, as it did not cross react with cytoplasmic actin of the nervous system. The molecular weight of the muscle actin in Corcyra was found to be around 46 kDa. The concentration of actin was found to be more or less the same in penultimate and early-last instar larvae and it increased during mid and late-last instar development. Thereafter, it has dropped to its minimum during the pupal stage. But, once again it increased to its maximum in adults. Thorax-ligation in early and late-last instar larvae resulted in decreased synthesis of muscle actin and this was more pronounced after 48 h of ligation. Injection of 20hydroxyecdysone to insects which were thorax-ligated for 24 and 48 h was found to significantly increase the concentration of actin, suggesting that the ecdysteroids mediate the muscle actin synthesis during the larval development of Corcyra.

In vitro studies with the larval CNS of the silkworm, Bombyx mori revealed the phosphorylation of a 48-kDa protein, which was not dependent on cyclic nucleotides. Studies also revealed modest phosphorylation of this protein by a calcium-dependent but calmodulin-independent mechanism. However, phosphorylation of this protein was greatly enhanced in the presence of juvenile hormone (JH) I by a calcium-independent mechanism. This stimulatory effect of JH was seen in both homogenates as well as in intact CNS of Bombyx. Immunoblotting studies revealed the cross-reaction of this 48-kDa protein with phosphotyrosine monoclonal antibody and the phosphorylation of this protein was inhibited by genistein. This study suggests that the 48-kDa protein is a substrate for tyrosine kinase. The phosphorylation of this protein was also observed in other larval tissues such as salivary gland, fat body, and epidermis of Bombyx.