Genistein, a protein tyrosine kinase inhibitor, activates the cystic fibrosis transmembrane conductance regulator (CFTR) in transfected NIH-3T3 fibroblasts that express the CFTR (3T3-CFTR). CFTR activity was assayed by 125I efflux and by patch clamping in the cell-attached mode. Both forskolin and genistein stimulated 125I efflux and activated a 9-10 pS anion channel in 3T3-CFTR cells but failed to activate 125I efflux in mock-transfected NIH-3T3 cells. Genistein, unlike forskolin and 3-isobutyl-1-methylxanthine, did not increase intracellular adenosine 3',5'-cyclic monophosphate (cAMP) above control levels. This demonstrates that genistein-dependent activation does not involve inhibition of phosphodiesterase activity and suggests that stimulation does not involve a direct activation of protein kinase A. Genistein stimulated 125I efflux to approximately 50% of the maximal rate with forskolin. Genistein did not increase 125I efflux at saturating forskolin but decreased the concentration of forskolin required for half-maximal stimulation. Orthovanadate (VO4), a phosphotyrosine phosphatase inhibitor, inhibited genistein-induced channel activation with an inhibition constant of approximately 20 microM. These effects suggest that, in addition to activation by protein kinase A, the CFTR is regulated by a tyrosine kinase-dependent pathway.

The platelet membrane is lined with a membrane skeleton that associates with transmembrane adhesion receptors and is thought to play a role in regulating the stability of the membrane, distribution and function of adhesive receptors, and adhesive receptor-induced transmembrane signaling. When platelets are lysed with Triton X-100, cytoplasmic actin filaments can be sedimented by centrifugation at low g-forces (15,600 ؋ g) but the membrane skeleton requires 100,000 ؋ g. The present study shows that DRP (dystrophin-related protein) sediments from lysed platelets along with membrane skeleton proteins. Sedimentation results from association with the membrane skeleton because DRP was released into the detergent-soluble fraction when actin filaments were depolymerized. Interaction of fibrinogen with the integrin ␣ IIb ␤ 3 induces platelet aggregation, transmembrane signaling, and the formation of integrin-rich cytoskeletal complexes that can be sedimented from detergent lysates at low g-forces. Like other membrane skeleton proteins, DRP redistributed from the high-speed pellet to the integrin-rich low-speed pellet of aggregating platelets. One of the signaling enzymes that is activated following ␣ IIb ␤ 3 -ligand interactions in a platelet aggregate is calpain; DRP was cleaved by calpain to generate a ϳ140-kDa fragment that remained associated with the low-speed detergent-insoluble fraction. These studies show that DRP is part of the platelet membrane skeleton and indicate that DRP participates in the cytoskeletal reorganizations resulting from signal transmission between extracellular adhesive ligand and the interior of the cell.Duchenne muscular dystrophy is one of the most common inherited human diseases. It is caused by a defective gene that codes for a 427-kDa protein, dystrophin (1-5). The deduced amino acid sequence of dystrophin shows that it consists of four domains and suggests that it is a cytoskeletal protein (6). The major rod-shaped domain contains 24 spectrin-like repeats. This domain is flanked on the amino terminus by a domain that has a high degree of homology to the actin-binding domains of spectrin and ␣-actinin, and on the carboxyl terminus by a cysteine-rich domain that shows some homology to a Ca 2ϩ -binding region in ␣-actinin. The most carboxyl-terminal end of dystrophin consists of a short domain that has no homology to any known protein and appears to play a role in linking the molecule to the plasma membrane (7,8). Recent studies using purified protein or recombinant fragments containing the putative actin-binding domain (9 -11) have shown that the protein can bind to actin filaments in vitro, supporting the idea that this molecule functions as a cytoskeletal protein. The finding that dystrophin exists in a submembranous location (8,12,13) and that the carboxyl-terminal end of the molecule associates tightly with a complex of membrane glycoproteins (termed dystroglycan) (14 -17) suggests that dystrophin is a component of a submembranous cytoskeleton.Although there is now considerable inform...

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Testosterone is responsible for several changes in the brain, including behavioral and emotional responses, memory, and cognition. Given this, we investigated changes in the brain wave profile caused by supplementation with exogenous testosterone in both castrated and non-castrated rats. We also investigated the serum testosterone levels, renal and hepatic function, and the lipid and behavioral profiles. We found changes in the spectral wave power in both groups (castrated and non-castrated animals) supplemented with exogenous testosterone, consistent with an aggressive/hostile profile. These changes were observed in the electrocorticographic evaluation associated with increased power in low-frequency (delta and theta) and high-frequency (beta and gamma) activity in the supplemented animals. The castrated animals presented a significant decrease of wave power in the alpha frequency. This correlated with a decrease of the performance of the animals in the elevated plus-maze evaluation, given that the alpha wave is linked to the execution and visualization of motor processes. In the behavioral evaluation, the castrated animals presented a reduced permanence time in the elevated-plus maze, although this was prevented by the supplementation of testosterone. Testosterone supplementation induced aggressive behavior in non-castrated animals, but not in castrated ones. Supplemented animals had significantly elevated serum testosterone levels, while their urea levels were significantly lower, but without clinical significance. Our data indicate that testosterone supplementation in non-castrated rats, but not in castrated ones, causes electrocorticographic changes that could be associated with more aggressive and hostile behavior, in addition to indicating a potential for personality disorder. However, further studies are required to elucidate the cellular and molecular changes caused by acute testosterone supplementation.

Short-term cultures of normal human mammary epithelial cells were used to determine the extent to which c-myc, c-Ha-ras1, and c-erbB-2 proto-oncogenes were expressed in proliferating normal cells. This level of expression was compared with that of primary tumor cells, malignant effusion cells, or permanently established breast cancer cell lines. Pure preparations of epithelial organoids from seven different reduction mammoplasty tissue samples yielded proliferating normal epithelial cells upon short-term tissue culture. In every sample, proto-oncogene transcript levels increased upon short-term culture of the epithelial cells. These levels often exceeded by 10-fold the levels measured in uncultured organoids from the same tissue. In four of the seven cultured normal breast samples, at least one of the proto-oncogenes increased its expression to a level equaling or exceeding that found in a proliferating breast cancer cell line, MCF7. One effusion metastasis sample and two primary ductal adenocarcinomas were also examined for proto-oncogene expression. The effusion metastasis sample expressed high levels of c-erbB-2 messenger RNA, in accord with its amplified gene copy number; otherwise, the levels of proto-oncogene transcripts were low in unprocessed tumor and uncultured organoids, but they increased with proliferation of the tumor cells in culture. These results indicate that the variable expression of these proto-oncogenes observed in breast biopsy specimens needs to be controlled for cellular growth rate or proliferation index. Furthermore, these findings suggest that dysregulated proto-oncogene expression, rather than overexpression per se, needs to be evaluated as a possible mechanism contributing to the development of human breast cancer.

Introduction Local anesthetics are widely used in clinical practice. While toxicity is rare, these drugs can cause potentially lethal seizures. Objective In the present study, we investigated the electrocorticographic (ECoG) and electromyographic patterns of seizures induced by acute lidocaine (LA) toxicity and treated with anticonvulsant drugs. The study used adult male Wistar rats to describe of the seizure‐related behavior of LA and investigated the treatment with anticonvulsant drugs. Results The use of LA resulted in clear changes in the ECoG pattern, which presented characteristics of Status epilepticus, with increased intensity in all brainwaves. The decomposition of the cerebral waves showed an increase in the beta and gamma waves that may be related to tonic–clonic seizure. Although the treatment with anticonvulsants drugs reduces the power of brainwaves at frequencies between 1 and 40 Hz compared to the LA group, but only diazepam (DZP) was able to decrease the intensity of oscillations. The muscle contraction power also indicated a difference in the effectiveness of the three treatments. Conclusion The sum of the evidence indicates that LA causes status epilepticus and that DZP is the most effective treatment for the control of these seizures, by restoring the systemic values to levels close to those recorded in the control group.

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