Low-level sequence variation in Toxoplasma gondii calcium-dependent protein kinases among different genotypes.

The causative agent of toxoplasmosis, Toxoplasma gondii, can infect virtually all nucleated cell types of warm-blooded animals. In this study, we examined the sequence variation in calcium-dependent protein kinase 2 (CDPK2) genes among 13 T. gondii strains from different hosts and geographical locations. The results showed that the lengths of the complete CDPK2 DNA and cDNA sequences were 3671-3673 and 2136 bp, respectively, and the sequence variation was 0-0.9% among different T. gondii strains. Phylogenetic analysis based on the CDPK2 gene sequences revealed that T. gondii strains of the same genotypes were clustered in different clades. Further analysis of all the other T. gondii CDPK genes in genotype I (GT1), II (ME49), or III (VEG) strains indicated the T. gondii CDPK gene family is quite conserved, with sequence variation ranging from 0 to 1.40%. We concluded that CDPK2 as well as all the other CDPK genes in T. gondii cannot be used as proper markers for studying the variants of different T. gondii genotypes from different hosts and geographical locations, but their sequence conservation may be a useful feature promoting them as anti-T. gondii vaccine candidates in further studies.

The role of 5-HT2C receptors in touchscreen visual reversal learning in the rat: a cross-site study.

RATIONALE: Reversal learning requires associative learning and executive functioning to suppress non-adaptive responding. Reversal-learning deficits are observed in e.g. schizophrenia and obsessive-compulsive disorder and implicate neural circuitry including the orbitofrontal cortex (OFC). Serotonergic function has been strongly linked to visual reversal learning in humans and experimental animals but less is known about which receptor subtypes are involved. OBJECTIVES: The objectives of the study were to test the effects of systemic and intra-OFC 5-HT2C-receptor antagonism on visual reversal learning in rats and assess the psychological mechanisms underlying these effects within novel touchscreen paradigms. METHODS: In experiments 1-2, we used a novel 3-stimulus task to investigate the effects of 5-HT2C-receptor antagonism through SB 242084 (0.1, 0.5 and 1.0 mg/kg i.p.) cross-site. Experiment 3 assessed the effects of SB 242084 in 2-choice reversal learning. In experiment 4, we validated a novel touchscreen serial visual reversal task suitable for neuropharmacological microinfusions by baclofen-/muscimol-induced OFC inactivation. In experiment 5, we tested the effect of intra-OFC SB 242084 (1.0 or 3.0 µg/side) on performance in this task. RESULTS: In experiments 1-3, SB 242084 reduced early errors but increased late errors to criterion. In experiment 5, intra-OFC SB 242084 reduced early errors without increasing late errors in a reversal paradigm validated as OFC dependent (experiment 4). CONCLUSION: Intra-OFC 5-HT2C-receptor antagonism decreases perseveration in novel touchscreen reversal-learning paradigms for the rat. Systemic 5-HT2C-receptor antagonism additionally impairs late learning-a robust effect observed cross-site and potentially linked to impulsivity. These conclusions are discussed in terms of neural mechanisms underlying reversal learning and their relevance to psychiatric disorders.

Characterization of the Toxoplasma gondii hsp60 gene sequences from different hosts and geographical locations.

The intracellular protozoan Toxoplasma gondii is one of the most successful parasites, with the ability to invade all warm-blooded animals, including humans. T. gondii heat shock protein 60 (TgHSP60) plays an important role in intracellular survival and in the differentiation of the parasite, and is also recognized as being associated with its virulence. In the present study, we examined sequence variation in the hsp60 coding region among five T. gondii isolates from different hosts and geographical regions, which were compared with the corresponding sequences of strains ME49, 76K, and GT1 available in the ToxoDB databases. The length of the T. gondii hsp60 sequence was 1728 bp for all strains, and the A+T content ranged from 41.96 to 42.13%. The sequence alignment of the 8 T. gondii strains identified 20 variable positions (0-1.44%) and showed 1.16% overall sequence variation, suggesting a relatively considerable sequence diversity. Phylogenetic analysis of hsp60 sequences using Bayesian inference and maximum parsimony differentiated the two major clonal lineage types into their respective clusters, and thus separated atypical strains from classical genotypes. The results of the present study suggested that the coding region of the hsp60 gene may represent a novel genetic marker for intraspecies phylogenetic analyses of T. gondii.

FGF2 regulates melanocytes viability through the STAT3-transactivated PAX3 transcription.

PAX3 (paired box 3) is known to have an important role in melanocyte development through modulation of microphthalmia-associated transcription factor transcription. Here we found that PAX3 transcriptional activity could be regulated through FGF2 (basic fibroblast growth factor)-STAT3 (signal transducer and activator of transcription 3) signaling in the pigment cells. To study its function in vivo, we have generated a transgenic mouse model expressing PAX3 driven by tyrosinase promoter in a tissue-specific fashion. These animals exhibit hyperpigmentation in the epidermis, evident in the skin color of their ears and tails. We showed that the darker skin color results from both increased melanocyte numbers and melanin synthesis. Together, our study delineated a novel pathway in the melanocyte lineage, linking FGF2-STAT3 signaling to increased PAX3 transcription. Moreover, our results suggest that this pathway might contribute to the regulation of melanocyte numbers and melanin levels, and thereby provide an alternative strategy to induce pigmentation.

PAK1 hyperactivation is sufficient for mammary gland tumor formation.

Emerging data suggest that p21-activated kinase 1 (Pak1), a downstream signaling molecule of the small GTPases, growth factors, and lipid signaling, is upregulated or hyperactivated in human breast cancer. Until now, however, no direct causative role had been found for Pak1 in mammary tumor formation. We therefore sought to identify the role that Pak1 plays in mammary gland tumorigenesis. Our results showed that in a transgenic mouse model, overexpression of catalytically active Pak1 leads to the development of malignant mammary tumors and to a variety of other breast lesions, including focal solid nodules, ductal hyperplasia, and mini-intraductal neoplasm and adenoma. We also found that Pak1 hyperactivation increases the stimulation of downstream proliferative signaling effectors MEK1/2 and p38-MAPK in mammary tumor epithelial cells. Moreover, in our study, we detected expression of estrogen receptor-alpha expression and progesterone receptor expression during early stages of the lesions, but their expression was lost during the cells' transition to malignant invasive tumors. Finally, we found that consistent with a role in breast tumor progression, Pak1 expression and its nuclear accumulation was increased progressively during the transition from ductal hyperplasia to ductal carcinoma in situ to adenocarcinoma in widely used multistep polyoma-middle T-antigen transgenic mice. Together, these findings provide the first direct evidence that Pak1 deregulation may be sufficient for the formation of mammary gland tumors.

Molecular cloning and characterization of PELP1, a novel human coregulator of estrogen receptor alpha.

Nuclear hormone receptors (NRs) are transcription factors whose activity is regulated by ligands and by coactivators or corepressors. We report the characterization of a new NR coregulator: proline-, glutamic acid-, leucine-rich protein 1 (PELP1), a novel human protein that comprises 1,282 amino acids and is localized on chromosome 17. The primary structure of PELP1 consists of several motifs present in most transcriptional regulators including nine NR-interacting boxes (LXXLL motifs), a zinc finger, and glutamic acid- and proline-rich regions. We demonstrate that PELP1 is a coactivator of estrogen receptor alpha (ERalpha). PELP1 enhances 17beta-estradiol-dependent transcriptional activation from the estrogen response element in a dose-dependent manner. PELP1 interacts with ERalpha and also with general transcriptional coactivators p300 and cAMP response element-binding protein-binding protein. PELP1 was differentially expressed in various human and murine tissues with the highest expression levels in the testes, mammary glands, and brain. We also provide evidence supporting the developmental regulation of PELP1 expression in murine mammary glands, the detectable expression of PELP1 in human mammary cancer cell lines, and the enhanced expression of PELP1 in human breast tumors. These findings suggest that PELP1 is a novel coregulator of ERalpha and may have a role in breast cancer tumorigenesis.

Etk/Bmx tyrosine kinase activates Pak1 and regulates tumorigenicity of breast cancer cells.

Etk/Bmx, a member of the Tec family of nonreceptor protein-tyrosine kinases, is characterized by an N-terminal pleckstrin homology domain and has been shown to be a downstream effector of phosphatidylinositol 3-kinase. P21-activated kinase 1 (Pak1), another well characterized effector of phosphatidylinositol 3-kinase, has been implicated in the progression of breast cancer cells. In this study, we characterized the role of Etk in mammary development and tumorigenesis and explored the functional interactions between Etk and Pak1. We report that Etk expression is developmentally regulated in the mammary gland. Using transient transfection, coimmunoprecipitation and glutathione S-transferase-pull down assays, we showed that Etk directly associates with Pak1 via its N-terminal pleckstrin homology domain and also phosphorylates Pak1 on tyrosine residues. The expression of wild-type Etk in a non-invasive human breast cancer MCF-7 cells significantly increased proliferation and anchorage-independent growth of epithelial cancer cells. Conversely, expression of kinase-inactive mutant Etk-KQ suppressed the proliferation, anchorage-independent growth, and tumorigenicity of human breast cancer MDA-MB435 cells. These results indicate that Pak1 is a target of Etk and that Etk controls the proliferation as well as the anchorage-independent and tumorigenic growth of mammary epithelial cancer cells.

Transcriptional repression of oestrogen receptor by metastasis-associated protein 1 corepressor.

Activation of the heregulin/HER2 pathway in oestrogen receptor (ER)-positive breast-cancer cells leads to suppression of oestrogen-receptor element (ERE)-driven transcription and disruption of oestradiol responsiveness, and thus contributes to progression of tumours to more invasive phenotypes. Here we report the identification of metastatic-associated protein 1 (MTA1), a component of histone deacetylase (HDAC) and nucleosome-remodelling complexes, as a gene product induced by heregulin-beta1 (HRG). Stimulation of cells with HRG is accompanied by suppression of histone acetylation and enhancement of deacetylase activity. MTA1 is also a potent corepressor of ERE transcription, as it blocks the ability of oestradiol to stimulate ER-mediated transcription. The histone-deacetylase inhibitor trichostatin A blocks MTA1-mediated repression of ERE transcription. Furthermore, MTA1 directly interacts with histone deacetylase-1 and -2 and with the activation domain of ER-alpha. Overexpression of MTA1 in breast-cancer cells is accompanied by enhancement of the ability of cells to invade and to grow in an anchorage-independent manner. HRG also promotes interaction of MTA1 with endogenous ER and association of MTA1 or HDAC with ERE-responsive target-gene promoters in vivo. These results identify ER-mediated transcription as a nuclear target of MTA1 and indicate that HDAC complexes associated with the MTA1 corepressor may mediate ER transcriptional repression by HRG.

Growth factors regulate heterogeneous nuclear ribonucleoprotein K expression and function.

Epidermal growth factor (EGF) family of growth factors and their receptors regulate normal and cancerous epithelial cell proliferation, a process that can be suppressed by antireceptor blocking antibodies. To identify genes whose expression may be modulated by antireceptor blocking antibodies, we performed a differential display screen with cells grown in the presence or absence of antireceptor blocking antibodies; isolates from one cDNA clone were 100% identical to human heterogeneous nuclear ribonucleoprotein K (hnRNP K), a protein with a conserved KH motif and RGG boxes, has been implicated in such functions as sequence-specific DNA binding, transcription, RNA binding, and nucleocytoplasmic shuttling. Both EGF and heregulin-beta1 induced expression of hnRNP K mRNA and protein in human breast cancer cells. This growth factor-mediated hnRNP K expression was effectively blocked by pretreatment of cultures with humanized anti-EGF receptor (EGFR) antibody C225, or anti-human epidermal growth factor receptor-2 (HER2) antibody. Anti-EGFR monoclonal antibody also caused regression of human tumor xenografts and reduction in hnRNP K levels in athymic mice. Samples from grade III human breast cancer contained more hnRNP K protein than samples from grade II cancer. Finally, overexpression of hnRNP K in breast cancer cells significantly increased target c-myc promoter activity and c-Myc protein, hnRNP K protein levels, and enhanced breast cancer cell proliferation and growth in an anchorage-independent manner. These results suggested that the activity of human EGF receptor family members regulates hnRNP K expression by extracellular growth promoting signals and that therapeutic humanized antibodies against EGFR and HER2 can effectively block this function.

[Analysis of soybean lecithin by supercritical fluid chromatography].

Separation of six phospholipids, phosphatidyl choline (PC), phosphatidyl-ethanolamine (PE), phosphatidyl-serine(PS), phosphatidyl-inositol(PI), phosphotidic acid(PA), lyso-phosphatidyl-choline(lyso-PC), in soybean lecithin with supercritical fluid chromatography was achieved within 15 min. C18 column was used and carbon dioxide modified by ethanol containing 0.05% (V/V) triethylamine was chosen as the mobile phase. Effects of the composition of mobile phase, temperature and pressure were studied. The quantitative analysis of PC has been achieved with external standard method. The calibration curve for PC was linear in the range between 0.020 g/L-0.075 g/L and the detection limit was 0.2 microgram. This method has been applied to the analysis of PC in soybean lecithin.

Evidence of Rab3A expression, regulation of vesicle trafficking, and cellular secretion in response to heregulin in mammary epithelial cells.

Heregulin beta1 (HRG), a combinatorial ligand for human growth factor receptors 3 and 4, is a regulatory polypeptide that promotes the differentiation of mammary epithelial cells into secretory lobuloalveoli. Emerging evidence suggests that the processes of secretory pathways, such as biogenesis and trafficking of vesicles in neurons and adipose cells, are regulated by the Rab family of low-molecular-weight GTPases. In this study, we identified Rab3A as a gene product induced by HRG. Full-length Rab3A was cloned from a mammary gland cDNA library. We demonstrated that HRG stimulation of human breast cancer cells and normal breast epithelial cells induces the expression of Rab3A protein and mRNA in a cycloheximide-independent manner. HRG-mediated induction of Rab3A expression was blocked by an inhibitor of phosphatidylinositol 3-kinase but not by inhibitors of mitogen-activated protein kinases p38(MAPK) and p42/44(MAPK). Human breast epithelial cells also express other components of regulated vesicular traffic, such as rabphilin 3A, Doc2, and syntaxin. Rab3A was predominantly localized in the cytosol, and HRG stimulation of the epithelial cells also raised the level of membrane-bound Rab3A. HRG treatment induced a profound alteration in the cell morphology in which cells displayed neuron-like membrane extensions that contained Rab3A-coated, vesicle-like structures. In addition, HRG also promoted the secretion of cellular proteins from the mammary epithelial cells. The ability of HRG to modify exocytosis was verified by using a growth hormone transient-transfection system. Analysis of mouse mammary gland development revealed the expression of Rab3A in mammary epithelial cells. Furthermore, expression of the HRG transgene in Harderian tumors in mice also enhanced the expression of Rab3A. These observations provide new evidence of the existence of a Rab3A pathway in mammary epithelial cells and suggest that it may play a role in vesicle trafficking and secretion of proteins from epithelial cells in response to stimulation by the HRG expressed within the mammary mesenchyma.

Vascular endothelial growth factor up-regulation via p21-activated kinase-1 signaling regulates heregulin-beta1-mediated angiogenesis.

Heregulin-beta1 promotes the activation of p21-activated kinase 1 (Pak1) and the motility and invasiveness of breast cancer cells. In this study, we identified vascular endothelial growth factor (VEGF) as a gene product induced by heregulin-beta1. The stimulation by heregulin-beta1 of breast cancer epithelial cells induced the expression of the VEGF mRNA and protein and its promoter activity. Heregulin-beta1 also stimulated angiogenesis in a VEGF-dependent manner. Herceptin, an anti-HER2 antibody inhibited heregulin-beta1-mediated stimulation of both VEGF expression in epithelial cells and angiogenesis in endothelial cells. Because the activation of Pak1 and VEGF expression are positively regulated by heregulin-beta1, we hypothesized that Pak1 regulates VEGF expression, and hence explored the role of Pak1 in angiogenesis. We provide new evidence to implicate Pak1 signaling in VEGF expression. Overexpression of a kinase-dead K299R Pak1 leads to suppression of VEGF promoter activity, as well as VEGF mRNA expression and secretion of VEGF protein. Conversely, kinase-active T423E Pak1 promotes the expression and secretion of VEGF. Furthermore, expression of the heregulin-beta1 transgene, HRG, in harderian tumors in mice enhances the activation of Pak1 as well as expression of VEGF and angiogenic marker CD34 antigen. These results suggest that heregulin-beta1 regulates angiogenesis via up-regulation of VEGF expression and that Pak1 plays an important role in controlling VEGF expression and, consequently, VEGF secretion and function.

Regulatable expression of p21-activated kinase-1 promotes anchorage-independent growth and abnormal organization of mitotic spindles in human epithelial breast cancer cells.

Stimulation of growth factor signaling has been implicated in the development of invasive phenotypes and the activation of p21-activated kinase (Pak1) in human breast cancer cells (Adam, L., Vadlamudi, R., Kondapaka, S. B., Chernoff, J., Mendelsohn, J., and Kumar, R. (1998) J. Biol. Chem. 273, 28238-28246; Adam, L., Vadlamudi, R., Mandal, M., Chernoff, J., and Kumar, R. (2000) J. Biol. Chem. 275, 12041-12050). To study the role of Pak1 in the regulation of motility and growth of breast epithelial cells, we developed human epithelial MCF-7 clones that overexpressed the kinase-active T423E Pak1 mutant under an inducible tetracycline promoter or that stably expressed the kinase-active H83L,H86L Pak1 mutant, which is deficient in small GTPase binding sites. The expression of both T423E and H83L,H86L Pak1 mutants in breast epithelial cells was accompanied by increased cell motility without any apparent effect on the growth rate of cells. The T423E Pak1 mutant was primarily localized to filopodia, and the H83L,H86L Pak1 mutant was primarily localized to ruffles. Cells expressing T423E Pak1 exhibited a regulatable stimulation of mitogen-activated protein kinase and Jun N-terminal kinase activities. The expression of kinase-active Pak1 mutants significantly stimulated anchorage-independent growth of cells in soft agar in a preferential mitogen-activated protein kinase-sensitive manner. In addition, regulatable expression of kinase-active Pak1 resulted in an abnormal organization of mitotic spindles characterized by appearance of multiple spindle orientations. We also provide evidence to suggest a close correlation between the status of Pak1 kinase activity and base-line invasiveness of human breast cancer cells and breast tumor grades. This study is the first demonstration of Pak1 regulation of anchorage-independent growth, potential Pak1 regulation of invasiveness, and abnormal organization of mitotic spindles of human epithelial breast cancer cells.

Transforming growth factor beta signal transducer Smad2 is expressed in mouse meiotic germ cells, Sertoli cells, and Leydig cells during spermatogenesis.

Although previous studies have shown that members of the transforming growth factor beta (TGFbeta) family are expressed in the seminiferous tubules, the functions of these growth factors in spermatogenesis remain elusive. In order to shed light on the mechanisms of TGFbeta action in spermatogenesis, it is crucial to determine whether and where their downstream signaling molecules are expressed in the testis. We examined the expression of Smad2, an intracellular signal transducer of the TGFbetas, in mouse testes by in situ hybridization and immunohistochemistry. Both Smad2 mRNA and protein were detected in meiotic germ cells, from preleptotene to pachytene spermatocytes, but not in postmeiotic germ cells. Smad2 expression was also observed in interstitial cells and Sertoli cells. Therefore, our data provide molecular evidence for TGFbeta signal transduction during spermatogenesis.

Autonomous cell death of mouse male germ cells during fetal and postnatal period.

Germ cell degeneration is common in mammalian testes during the developmental as well as the adult period. To investigate the extent and mechanisms of male germ cell death during fetal and neonatal life, the testes of mice at various fetal and postnatal ages extending from 13 days of gestation to 7 wk after birth were examined by electron microscopy and/or terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL). Electron microscopy revealed that the number of cells with typical features of spermatogenic cell apoptosis was highest at 13 days of gestation, coinciding with the time of immigration of primordial germ cells into gonads. A second peak was observed around 10-13 days after birth when the first wave of spermatogenesis had started and active spermatogonial proliferation was present. Surprisingly, we found a significant number of dying cells around birth, which exhibited morphological features of necrotic death. In agreement with the results of electron microscopy, TUNEL staining revealed that the dying germ cells present around birth were TUNEL negative, while positive nuclei were abundant in the lumen of seminiferous tubules of testes of 10- to 13-day-old mice. To investigate the mechanisms of induction of germ cell death, we examined the expression of Fas antigen immunohistochemically using rabbit antiserum raised against synthetic peptides for part of mouse Fas antigen. We found that among various developmental stages investigated, positive immunostaining for Fas antigen was present between 17 days of gestation and 1 day after birth, with the most intensive staining occurring on 17 days of gestation. Therefore, Fas-induced pathways may be implicated in embryonic male germ cell death, not prepubertal spermatogenic cell death.

alpha-subunit of CaM-KII increases glycine currents in acutely isolated rat spinal neurons.

1. Here we report that in acutely isolated rat spinal dorsal horn neurons, the glycine receptor can be regulated by exogenous calcium/ calmodulin-dependent protein kinase II (CaM-KII). Intracellularly applied, the alpha-subunit of CaM-KII enhanced glycine receptor-activated current recorded with the use of the whole cell patch-clamp technique. This result suggests that the function of glycine receptor is modulated by CaM-KII, but the cellular mechanism underlying the enhancement of glycine receptor-activated current is still unknown.

Alpha-subunit of calcium/calmodulin-dependent protein kinase II enhances gamma-aminobutyric acid and inhibitory synaptic responses of rat neurons in vitro.

1. Here we report that in acutely isolated rat spinal dorsal horn neurons, the gamma-aminobutyric acid-A (GABAA) receptor can be regulated by calcium/calmodulin-dependent protein kinase II (CaM-KII). Intracellularly applied, the alpha-subunit of CaM-KII enhanced GABAA-receptor-activated current recorded with the use of the whole cell patch-clamp technique. This effect was associated with reduced desensitization of GABA responses. 2. GABA-induced currents are also potentiated by calyculin A, an inhibitor of protein phosphatases 1 and 2A. 3. Conventional intracellular recordings were made from hippocampal CA1 neurons in slices to determine the effect of intracellular application of CaM-KII on inhibitory synaptic potentials evoked by electrical stimulation of the stratum oriens/alveus. The inhibitory synaptic potential was enhanced by CaM-KII; this mechanism may contribute to long-term enhancement of inhibitory synaptic transmission and may also play a role in other forms of plasticity in the mammalian brain.

Activation of mu-opioid receptor modulates GABAA receptor-mediated currents in isolated spinal dorsal horn neurons.

Whole-cell voltage-clamp technique was used to examine the effects of a mu-opioid receptor agonist DAGO (Tyr-D-Ala-Gly-Me-Phe-Gly-ol-enkephalin) on GABA-induced currents in acutely isolated spinal dorsal horn (DH) neurons from laminae I-IV of young rats. We found that a bicuculline-sensitive GABA-induced current was potentiated by DAGO (0.5-500 nM), in a dose-dependent manner, in approximately 62% of the tested cells. The elevated GABA responses outlasted the period of DAGO application, and either recovered within 10 min after the removal of the peptide or persisted for up to 50 min. The potentiating effect of DAGO was reduced or prevented by naloxone and the mu-opioid receptor-selective antagonist beta-funaltrexamine. A similar enhancing effect on the membrane currents activated by administration of muscimol, a GABAA receptor-specific agonist, was produced by DAGO. In addition, a transient depression of GABA responses was observed in approximately 25% of the cells tested. These results indicate that the mu-opioid agonist DAGO modulates the sensitivity of postsynaptic GABAA receptors in a large proportion of spinal neurons from laminae I-IV, with the major effect being facilitation. The DAGO action could contribute to the regulation of the strength of primary afferent neurotransmission, including nociception.