Modulation of NMDA receptors by pituitary adenylate cyclase activating peptide in CA1 neurons requires G alpha q, protein kinase C, and activation of Src.

At CA1 synapses, activation of NMDA receptors (NMDARs) is required for the induction of both long-term potentiation and depression. The basal level of activity of these receptors is controlled by converging cell signals from G-protein-coupled receptors and receptor tyrosine kinases. Pituitary adenylate cyclase activating peptide (PACAP) is implicated in the regulation of synaptic plasticity because it enhances NMDAR responses by stimulating Galphas-coupled receptors and protein kinase A (Yaka et al., 2003). However, the major hippocampal PACAP1 receptor (PAC1R) also signals via Galphaq subunits and protein kinase C (PKC). In CA1 neurons, we showed that PACAP38 (1 nM) enhanced synaptic NMDA, and evoked NMDAR, currents in isolated CA1 neurons via activation of the PAC1R, Galphaq, and PKC. The signaling was blocked by intracellular applications of the Src inhibitory peptide Src(40-58). Immunoblots confirmed that PACAP38 biochemically activates Src. A Galphaq pathway is responsible for this Src-dependent PACAP enhancement because it was attenuated in mice lacking expression of phospholipase C beta1, it was blocked by preventing elevations in intracellular Ca2+, and it was eliminated by inhibiting either PKC or cell adhesion kinase beta [CAKbeta or Pyk2 (proline rich tyrosine kinase 2)]. Peptides that mimic the binding sites for either Fyn or Src on receptor for activated C kinase-1 (RACK1) also enhanced NMDAR in CA1 neurons, but their effects were blocked by Src(40-58), implying that Src is the ultimate regulator of NMDARs. RACK1 serves as a hub for PKC, Fyn, and Src and facilitates the regulation of basal NMDAR activity in CA1 hippocampal neurons.

Regulation of NMDA receptor activity by F-actin and myosin light chain kinase.

The postsynaptic density (PSD) at excitatory dendritic synapses comprises a protein complex of glutamate receptors, scaffolding elements, and signaling enzymes. For example, NMDA receptors (NMDARs) are linked to several proteins in the PSD, such as PSD-95, and are also tethered via binding proteins such as alpha-actinin directly to filamentous actin of the cytoskeleton. Depolymerization of the cytoskeleton modulates the activity of NMDARs, and, in turn, strong activation of NMDARs can trigger depolymerization of actin. Myosin, the motor protein of muscular contraction and nonmuscle motility, is also associated with NMDARs and the PSD. We show here that constitutively active myosin light chain kinase (MLCK) enhances NMDAR-mediated whole-cell and synaptic currents in acutely isolated CA1 pyramidal and cultured hippocampal neurons, whereas inhibitors of MLCK depress these currents. This MLCK-dependent regulation was observed in cell-attached patches but was lost after excision to inside-out patches. Furthermore, the enhancement induced by constitutively active MLCK and the depression of MLCK inhibitors were eliminated after depolymerization of the cytoskeleton. NMDARs and MLCK did not colocalize in clusters on the dendrites of cultured hippocampal neurons, further indicating that the effects of MLCK are mediated indirectly via actomyosin. Our results suggest that MLCK enhances actomyosin contractility to either increase the membrane tension on NMDARs or to alter physical relationships between the actin cytoskeleton and the linker proteins of NMDARs.

Impaired steroidogenic factor 1 (NR5A1) activity in mutant Y1 mouse adrenocortical tumor cells.

Mutants isolated from the Y1 mouse adrenocortical tumor cell line (clones 10r-9 and 10r-6) are resistant to ACTH because they fail to express the melanocortin-2 receptor (MC2R). In this study, we show that a luciferase reporter plasmid driven by 1,800 bp of the proximal promoter region of the MC2R was expressed poorly in the mutant cells compared with parent Y1 cells. The differential expression of the MC2R in parent and mutant cells resulted from impaired activity of the orphan nuclear receptor NR5A1 (SF1) on the promoter as determined by 5'-deletion analysis. Furthermore, the activity of an SF1 expression plasmid on an SF1-dependent reporter plasmid was compromised in mutant clones. The site-specific DNA binding properties of SF1 from parent and mutant cells did not differ as determined in electrophoretic mobility shift assays, and the addition of the activation domain of VP16 to the amino terminus of SF1 restored the transcriptional activity of the protein. In addition, the levels of SF1 and other cofactors including WT1, CBP/p300, and steroid receptor coactivator 1 did not differ appreciably between parent and mutant cells. Taken together, these results suggest that ACTH resistance in the mutant clones resulted from a defect that affected the activation properties of SF1 rather than its DNA binding activity. Consistent with the observed impairment in SF1 function, other SF1-dependent genes, including Cyp11b1 and steroidogenic acute regulatory protein (StAR), were poorly expressed and global steroidogenesis, as evidenced by the metabolism of 22(R)-hydroxycholesterol to steroid products, was impaired. Interestingly, MC2R, Cyp11a, Cyp11b1, and StAR transcripts were not affected to the same degree, suggesting that each of these genes may have a different absolute requirement for SF1. These mutants thus provide an experimental paradigm to identify factors that influence SF1 function and to evaluate the relative importance of SF1 in the expression of genes essential for adrenal steroidogenesis.

Amphetamine-related disorders.

This is a review paper in which the author looks at amphetamine, its origin, place in history, epidemiologic data, mechanism of action, detection methods, new approaches in withdrawal, its toxicity in laboratory animals, use, and clinically observed results.

Altered G protein activity in a desensitization-resistant mutant of the Y1 adrenocortical tumor cell line.

Mutant isolates [designated desensitization resistant (DR)] from the Y1 mouse adrenocortical tumor cell line resist agonist-induced desensitization of adenylyl cyclase by preventing the uncoupling of receptors from their guanyl nucleotide-binding regulatory G proteins. In this study, we tested the hypothesis that an underlying G protein defect is associated with the DR phenotype. We found that the G protein reagent guanyl-5'-yl imidodiphosphate [Gpp(NH)p] shifted beta2-adrenergic receptors from a high affinity state to a low affinity state 4-fold more effectively in mutant DR cells than in parent Y1 cells. In the DR mutant, Gpp(NH)p was able to shift receptors to a low affinity state in the absence of NaCl, whereas the effect of Gpp(NH)p in parent Y1 cells was dependent upon the presence of NaCl. Moreover, these differences in sensitivity to Gpp(NH)p and NaCl were transferred to Gs alpha-deficient S49(CYC-) lymphoma cell membranes in G protein reconstitution assays. These observations suggested that the DR mutation was associated with altered activity of the stimulatory G protein, Gs. Cloning and sequence analysis demonstrated that Gs alpha transcripts in the DR mutant were normal, suggesting that another factor involved in guanyl nucleotide exchange is responsible for the altered G protein activity in DR mutant cells.

Amplification of the transketolase gene in desensitization-resistant mutant Y1 mouse adrenocortical tumor cells.

As shown previously, mutants of the Y1 mouse adrenocortical tumor cell line that resist agonist-induced desensitization of adenylyl cyclase have elevated levels of a 68-kDa protein (designated p68), suggesting a possible relationship between p68 and the regulation of adenylyl cyclase activity. In the present study, cDNA cloning and sequencing were used to identify p68 as mouse transketolase. Cells overexpressing p68 exhibited a 17.4-fold increase in transketolase enzymatic activity relative to parental Y1 cells and a 28-fold amplification of the transketolase gene as determined by Southern blot hybridization analysis. Using fluorescent in situ hybridization analysis, the transketolase gene was mapped to mouse chromosome 16B1 and to human chromosome 3p21.2. Transketolase gene amplification was associated with telomeric fusion of the chromosome 16 pair together with the appearance of multiple copies of the transketolase gene throughout a different chromosome. The relationship between overexpression of transketolase and desensitization resistance was evaluated in somatic cell hybrids formed between a desensitization-resistant adrenal cell line and a desensitization-sensitive rat glial cell line. In these hybrids, transketolase overexpression behaved dominantly, whereas desensitization resistance behaved recessively. These results dissociate the desensitization resistance phenotype from overexpression of transketolase and suggest that desensitization resistance may have resulted from disruption of an essential regulatory gene in conjunction with the amplification event.

Diagnostic and prognostic significance of the mitotic index in endometrial adenocarcinoma.

The aim of current study was to evaluate the diagnostic as well as the prognostic significance of the mitotic index (MI) in endometrial adenocarcinoma. We compared the MI in normal endometrium, endometrial hyperplasia, and endometrial adenocarcinoma. The mean MI in normal proliferative endometrium (4.35 +/- 3.4) was not significantly different from those in glandular hyperplasia (4.19 +/- 6.0) and well-differentiated adenocarcinoma (4.01 +/- 4.2). A significantly higher MI (10.7 +/- 8.2) was found only in poorly differentiated adenocarcinoma (P less than 0.05). Results of our work indicate that the MI cannot be used as a discriminating factor in the differential diagnosis of borderline cases of endometrial hyperplasia and endometrial adenocarcinoma. We examined the usefulness of the MI, grade of differentiation, and depth of invasion as the prognostic factors in endometrial adenocarcinoma. The significantly higher 5-year mortality rate was associated with an MI greater than 5, grade III of differentiation, and neoplastic invasion penetrating to the outer third of the myometrium. All of the patients with MI greater than 5 had tumors with the highest grade of differentiation and/or invasion involving the mid and outer third of myometrium. This suggests that the higher mortality of patients with an MI greater than 5 reflects the presence of anaplastic and/or highly invasive tumor.

[Evaluation of the methods of treatment of children with liver neoplasms]. / Ocena metod leczenia dzieci z nowotworami watroby.

Unfavorable prognosis for children with malignant liver tumors, is caused not only because of late diagnosis in this children, but because of imperfet methods of treatment. These methods taken from adults oncology have to be adapted for pediatric patients in front of their differences in biology, pharmacodynamic reactivity and tumor-host relationships in developing organism. Some methods of treatment can be done the same way as in adults, but another (intraarterial treatment) can be used in children with much better results then in adults. Primary malignant tumors in children are hepatoblastoma and hepatocelullar carcinoma (minority) and metastatic tumors are nephroblastoma or neuroblastoma. That makes probably basic difference with liver tumors in adults, as well as absence of hepatic cirrhosis in children. 42 children with primary and metastatic liver tumors were treated by the author in Clinical Department of Pediatric Oncology, Institute of Mother and Child, Warsaw. 19 of them was given intra-arterial chemotherapy for unoperable primary tumors, 6--systemic chemotherapy for the same reason, 5--radiotherapy (all of them neuroblastomas) and 15 was submited to surgery-From this group 13 was operated radicaly by means of right extended or left lobectomy. Only in one case, middle lobectomy was done. 3 children operated radicaly was previously treated with intra-arterial chemotherapy and only after significant remission, surgery was done. As a result 6 children is RFS for more than 3 years. One of them exclusively grace to intra-arterial treatment. Experimental investigation done in monkeys shown no long term morphological and/or functional disturbances after long time hepatic infusion with chemotherapeutic drugs. In conclusion it is to state that surgery is the best method of treatment in primary liver tumors, some time preceded with intra-aterial chemotherapy that is safe for normal liver tissue. In metastatic neuroblastoma in liver, radiotherapy with systemic chemotherapy can give 60% of RFS (recurrent free survival).

[Preliminary elaboration of the method of complex treatment of rhabdomyosarcoma]. / Wstepne opracowanie metody leczenia kompleksowego nowotworów miesni poprzecznie prazkowanych.

24 children with rhabdomyosarcoma were treated in Clinical Department of Pediatric Oncology of the Institute of Mother and Child, Warsaw during last 2 years. Complex therapy consisted of surgery, chemotherapy, radiotherapy and rehabilitation. Choice method of the treatment depends of stagging by Intergroup RMS Study. Basic method is surgery (radical excision with margin of neighbouring tissue). Modern multi-drug chemotherapy has changed strategy of the treatment by means of less extended surgery. Radiotherapy in RMS has to be mega-voltage what gives short time of irradiation and less complication. Rehabilitation as a component of complex therapy of RMS has to preserve function of treated organ and in cases after mutilated surgery becomes rehabilitated procedure.

[Preliminary analysis and elaboration of the methods of study of the attitude of parents of children with neoplasms and the methods of psychotherapy and evaluation of physical fitness of these children]. / Wstepna analiza i opracowanie metod badania postaw rodziców dzieci z choroba nowotworowa oraz metod terapii psychologicznej i oceny sprawnosci ogólnej tych dzieci.

The main purpose of this paper is presentation of chosen methods of reintegration into main stream from the point of psychological, social development and physical capacity, in children with cancer. These methods concern to: investigation of parents' attitude towards children with cancer, group psychological therapy, evaluation of general physical capacity of the children and their locomotion function. There were done preliminary analysis of parents' attitude of children with cancer in diagnostic period. It consists a trial in verification of chosen methods.