Phorbol ester-stimulated phosphorylation of keratinocyte transglutaminase in the membrane anchorage region.

The membrane-bound transglutaminase of cultured keratinocytes became radioactively labelled upon addition of [32P]Pi to the medium. Transglutaminase phosphorylation was also demonstrable using particulate material isolated from cell homogenates. Compatible with mediation of the labelling by protein kinase C, the degree of phosphorylation in intact cells was stimulated approx. 5-fold in 4 h on treatment with the tumour-promoting phorbol ester phorbol 12-myristate 13-acetate, but not by phorbol. The extent of labelling was virtually unaffected by cycloheximide inhibition of protein synthesis, indicating that it arose primarily through turnover of phosphate in the membrane-bound enzyme. Phosphoamino acid analysis detected labelling only of serine residues. Most of the label was removed by trypsin release of the enzyme from the particulate fraction of cell homogenates, which deletes a membrane anchorage region of approximately 10 kDa. Upon trypsin treatment of the enzyme after immunoprecipitation, the phosphate label was recovered in soluble peptide material with a size of several thousand Da or less. Indicative of fragmentation of the membrane anchorage region, this material was separable by h.p.l.c. into two equally labelled peptides. Moreover, when the enzyme was labelled with [3H]palmitate or [3H]myristate, the fatty-acid-labelled peptide material required non-ionic detergent for solubilization and was separable from the phosphate-labelled material by gel filtration. Phorbol ester treatment of cultured keratinocytes in high- or low- Ca2(+)-containing medium was not accompanied by an appreciable protein-synthesis-independent change in transglutaminase activity. Independent of possible alteration of the intrinsic catalytic activity of the enzyme, phosphorylation may well modulate its interaction with substrate proteins, a potential site for physiological regulation.     

Acylation of keratinocyte transglutaminase by palmitic and myristic acids in the membrane Anchorage region

The membrane-bound form of keratinocyte transglutaminase was found to be labeled by addition of [3H] acetic, [3H]myristic, or [3H]palmitic acids to the culture medium of human epidermal cells. Acid methanolysis and high performance liquid chromatography analysis of palmitate-labeled transglutaminase yielded only methyl palmitate. In contrast, analysis of the myristate-labeled protein yielded approximately 40% methyl myristate and 60% methyl palmitate. Incorporation of neither label was significantly affected by cycloheximide inhibition of protein synthesis. The importance of the fatty acid moiety for membrane anchorage was demonstrated in three ways. First, the enzyme was solubilized from the particulate fraction of cell extracts by treatment with neutral 1 M hydroxylamine, which was sufficient to release the fatty acid label. Second, solubilization of active enzyme from the particulate fraction upon mild trypsin treatment resulted in a reduction in size by approximately 10 kDa and removal of the fatty acid radiolabels. Third, the small fraction of soluble transglutaminase in cell extracts was found almost completely to lack fatty acid labeling. Keratinocyte transglutaminase translated from poly(A+) RNA in a reticulocyte cell-free system was indistinguishable in size from the native enzyme, suggesting anchorage requires only minor post-translational processing. Thus, the data are highly compatible with membrane anchorage by means of fatty acid acylation within 10 kDa of the NH2 or COOH terminus.     

Piperidine-based heterocyclic oxalyl amides as potent p38α MAP kinase inhibitors

The design and synthesis of a new class of p38α MAP kinase inhibitors based on 4-fluorobenzylpiperidine heterocyclic oxalyl amides are described. Many of these compounds showed low-nanomolar activities in p38α enzymatic and cell-based cytokine TNFα production inhibition assays. The optimal linkers between the piperidine and the oxalyl amide were found to be [6,5] fused ring heterocycles. Substituted indoles and azaindoles were favored structural motifs in the cellular assay.     

Memory CD8+ T cell responses expand when antigen presentation overcomes T cell self-regulation

Antimicrobial memory CD8+ T cell responses are not readily expanded by either repeated infections or immunizations. This is a major obstacle to the development of T cell vaccines. Prime-boost immunization with heterologous microbes sharing the same CD8+ epitope can induce a large expansion of the CD8+ response; however, different vectors vary greatly in their ability to boost for reasons that are poorly understood. To investigate how efficient memory T cell expansion can occur, we evaluated immune regulatory events and Ag presentation after secondary immunization with strong and weak boosting vectors. We found that dendritic cells were essential for T cell boosting and that Ag presentation by these cells was regulated by cognate memory CD8+ T cells. When weak boosting vectors were used for secondary immunization, pre-established CD8+ T cells were able to effectively curtail Ag presentation, resulting in limited CD8+ T cell expansion. In contrast, a strong boosting vector, vaccinia virus, induced highly efficient Ag presentation that overcame regulation by cognate T cells and induced large numbers of memory CD8+ T cells to expand. Thus, efficient targeting of Ag to dendritic cells in the face of cognate immunity is an important requirement for T cell expansion.     

Synthesis and SAR of alpha-sulfonylcarboxylic acids as potent matrix metalloproteinase inhibitors

A series of novel carboxylic acid-based alpha-sulfone MMP inhibitors have been synthesized and the in vitro enzyme SAR is discussed. A potential binding mode in the active site of the MMP-9 homology model was highlighted. These compounds are potent MMP-9 inhibitors and are selective over MMP-1.     

Selective inhibition of p38alpha MAPK improves cardiac function and reduces myocardial apoptosis in rat model of myocardial injury

p38 MAPK is activated during heart diseases that might associate with myocardial damage and deterioration of cardiac function. In a rat model of myocardial injury, we have investigated cardioprotective effects of the inhibition of p38 MAPK using a novel, orally available p38alpha MAPK inhibitor. Rats were treated with N(omega)-nitro-l-arginine methyl ester (l-NAME, 40 mg.kg(-1).day(-1)) in drinking water plus 1% salt for 14 days and ANG II (0.5 mg.kg(-1).day(-1)) for 3 days. A selective p38alpha MAPK inhibitor, SD-282 (60 mg/kg), was administrated orally, twice a day for 4 days, starting 1 day before ANG II administration. The cardioprotective effects of p38alpha MAPK inhibition were evaluated by improvement of cardiac function, reduction of inflammatory cell infiltration, and cardiomyocyte apoptosis. SD-282 significantly improved cardiac function indicated by increasing stroke volume, cardiac output, ejection fraction, and stroke work and significantly decreasing arterial elastance. SD-282 also significantly reduced macrophage infiltration as judged by reduction of a specific marker, ED-1-positive staining cells (P < 0.05) in the myocardium. Furthermore, cardiomyocyte apoptosis as indicated by caspase-3 immunohistochemical staining was abolished by SD-282, and this effect may contribute to the reduction of myocardial damage evaluated by imaging analysis (P < 0.05 in both cases). Data suggest that p38alpha MAPK may play a critical role in the pathogenesis of cardiac dysfunction. Inhibition of p38alpha MAPK may be used as a novel cardioprotective strategy in attenuation of inflammatory response and deterioration of cardiac function that occurs in acute cardiovascular disease such as myocardial infarction.     

Growth factor regulation of estrogen receptor coregulator PELP1 functions via Protein Kinase A pathway

PELP1 (proline-rich, glutamic acid-rich, and leucine-rich protein-1) is a potential proto-oncogene that functions as a coregulator of estrogen receptor (ER), and its expression is deregulated during breast cancer progression. Emerging evidence suggests growth factor signaling crosstalk with ER as one possible mechanism by which breast tumors acquire resistance to therapy. In this study, we examined mechanisms by which growth factors modulate PELP1 functions, leading to activation of ER. Using in vivo labeling assays, we have found that growth factors promote phosphorylation of PELP1. Utilizing a panel of substrate-specific phosphorylated antibodies, we discovered that growth factor stimulation promotes phosphorylation of PELP1 that is recognized by a protein kinase A (PKA) substrate-specific antibody. Accordingly, growth factor-mediated PELP1 phosphorylation was effectively blocked by PKA-specific inhibitor H89. Utilizing purified PKA enzyme and in vitro kinase assays, we obtained evidence of direct PELP1 phosphorylation by PKA. Using deletion and mutational analysis, we identified PELP1 domains that are phosphorylated by PKA. Interestingly, site-directed mutagenesis of the putative PKA site in PELP1 compromised growth factor-induced activation and subnuclear localization of PELP1 and also affected PELP1-mediated transactivation function. Utilizing MCF-7 cells expressing a PELP1 mutant that cannot be phosphorylated by PKA, we provide mechanistic insights by which growth factor signaling regulates ER transactivation in a PELP1-dependent manner. Collectively, these findings suggest that growth factor signals promote phosphorylation of ER coactivator PELP1 via PKA pathway, and such modification may have functional implications in breast tumors with deregulated growth factor signaling.     

Multiple actions of spinophilin regulate mu opioid receptor function

Spinophilin, a dendritic spine-enriched scaffold protein, modulates synaptic transmission via multiple functions mediated by distinct domains of the protein. Here, we show that spinophilin is a key modulator of opiate action. Knockout of the spinophilin gene causes reduced sensitivity to the analgesic effects of morphine and early development of tolerance but a higher degree of physical dependence and increased sensitivity to the rewarding actions of the drug. At the cellular level, spinophilin associates with the mu opioid receptor (MOR) in striatum and modulates MOR signaling and endocytosis. Activation of MOR by opiate agonists such as fentanyl and morphine promotes these events, which feedback to suppress MOR responsiveness. Our findings support a potent physiological role of spinophilin in regulating MOR function and provide a potential new target for the treatment of opiate addiction.     

Exploration of the internal cavity of histone deacetylase (HDAC) with selective HDAC1/HDAC2 inhibitors (SHI-1:2)

We report herein the initial exploration of novel selective HDAC1/HDAC2 inhibitors (SHI-1:2). Optimized SHI-1:2 structures exhibit enhanced intrinsic activity against HDAC1 and HDAC2, and are greater than 100-fold selective versus other HDACs, including HDAC3. Based on the SAR of these agents and our current understanding of the HDAC active site, we postulate that the SHI-1:2 extend the existing HDAC inhibitor pharmacophore to include an internal binding domain.     

Oral dydrogesterone versus intravaginal micronised progesterone as luteal phase support in assisted reproductive technology (ART) cycles: results of a randomised study

The objective of this prospective, randomised study was to compare the efficacy, safety and tolerability of vaginal micronised progesterone with oral dydrogesterone as luteal phase support after in-vitro fertilisation (IVF). A total of 430 women underwent IVF/intracytoplasmic sperm injection (ICSI) treatment. Long protocol gonadotropin releasing hormone analogue down-regulation was followed by gonadotropin stimulation. Human chorionic gonadotropin was given when two or more follicles reached ≥17 mm. After 36 h, oocytes were retrieved and IVF was performed. Embryo transfer was done at the 4–8 cell embryo stage. Luteal support was initiated from the day of embryo transfer and continued for up to 14 days. Patients were randomised to luteal supplementation with either intravaginal micronised progesterone 200 mg three times daily (n = 351) or oral dydrogesterone 10 mg twice daily (n = 79). In cases of a positive pregnancy test, luteal support was continued for 12 weeks. Both dydrogesterone and micronised progesterone were associated with similar rates of successful pregnancies. Vaginal discharge or irritation were reported by 10.5% of patients given micronised progesterone. Significantly (p < 0.05), more patients given dydrogesterone than micronised progesterone were satisfied with the tolerability of their treatment. There were no differences between the treatments with regard to liver function tests.