The anti-apoptotic protein HAX-1 interacts with SERCA2 and regulates its protein levels to promote cell survival

Cardiac contractility is regulated through the activity of various key Ca²⁺-handling proteins. The sarco(endo)plasmic reticulum (SR) Ca²⁺ transport ATPase (SERCA2a) and its inhibitor phospholamban (PLN) control the uptake of Ca²⁺ by SR membranes during relaxation. Recently, the antiapoptotic HS-1–associated protein X-1 (HAX-1) was identified as a binding partner of PLN, and this interaction was postulated to regulate cell apoptosis. In the current study, we determined that HAX-1 can also bind to SERCA2. Deletion mapping analysis demonstrated that amino acid residues 575–594 of SERCA2's nucleotide binding domain are required for its interaction with the C-terminal domain of HAX-1, containing amino acids 203-245. In transiently cotransfected human embryonic kidney 293 cells, recombinant SERCA2 was specifically targeted to the ER, whereas HAX-1 selectively concentrated at mitochondria. On triple transfections with PLN, however, HAX-1 massively translocated to the ER membranes, where it codistributed with PLN and SERCA2. Overexpression of SERCA2 abrogated the protective effects of HAX-1 on cell survival, after hypoxia/reoxygenation or thapsigargin treatment. Importantly, HAX-1 overexpression was associated with down-regulation of SERCA2 expression levels, resulting in significant reduction of apparent ER Ca²⁺ levels. These findings suggest that HAX-1 may promote cell survival through modulation of SERCA2 protein levels and thus ER Ca²⁺ stores.     

Corticotropin-releasing hormone activates protein kinase C in an isoenzyme-specific manner

Protein kinase C (PKC) has recently emerged as mediator of corticotropin-releasing hormone (CRH) effects. Aim of the present study was to study the effects of CRH on each PKC isoenzyme. As a model we have used the PC12 rat pheochromocytoma cell line, expressing the CRH type 1 receptor (CRHR1). Our data were as follows: (a) CRH-induced rapid phosphorylation of conventional PKCalpha and PKCbeta, accompanied by parallel increase of their concentration within nucleus. (b) CRH suppressed the phosphorylation of novel PKCdelta and PKCtheta;, which remained in the cytosol. (c) CRH-induced transient phosphorylation of atypical PKClambda and had no effect on PKCmu. (d) The effect of CRH on each PKC isoenzyme was blocked by a CRHR1 antagonist. (e) Blockade of conventional PKC phosphorylation inhibited CRH-induced calcium ion mobilization from intracellular stores as well as the CRH-induced apoptosis and Fas ligand production. In conclusion, our findings suggest that CRH via its CRHR1 receptor differentially regulates PKC-isoenzyme phosphorylation, an apparently physiologically relevant effect since blockade of conventional PKC phosphorylation abolished the biological effect of CRH.     

Bioavailability of silymarin flavonolignans: drug formulations and biotransformation

Over the past years, great advances have been made on the development of novel delivery systems for bioactive natural compounds, in parallel to their structural modification via chemical, chemo-enzymatic and enzymatic methodologies. These approaches give rise to novel formulations and derivatives that often display advantages over the parental molecule, such as enhanced bioavailability and pharmacological activity, due to improved dissolution and stability. Silymarin components suffer from poor solubility in water and lipid media and their resorption in the intestine is rather limited. Moreover, silybin undergoes intensive Phase II metabolism and is rapidly excreted in bile and urine, leading to low therapeutic efficacy. This work aims to present the current status of available silymarin formulations, and to highlight successful efforts for the biotransformation of its constituent flavonolignans towards the synthesis of novel derivatives. Herein, various pharmaceutical formulations that aim at the bioavailability improvement of these fascinating phytochemicals, i.e., liposomes, phytosomes, self-microemulsifying drug delivery systems, solid dispersions systems, dripping pills, nanosuspensions, floating tablets, and micronization, are reviewed. Silybin (semi)synthetic derivatives prepared by chemical or enzymatic methods, such as fatty acid conjugates, silybin bishemisuccinate, silybin glycosides, silybin sulfates, silybinic acid, and 2,3-dehydrosilybin, are also discussed in detail. Additionally, this work attempts to direct the attention towards the pharmacological implications of optically pure silybin A and silybin B and their biotransformation reactions, both Phase I and II, in relation to bioavailability.     

Thrombin inhibitors with lipid peroxidation and lipoxygenase inhibitory activities

Vascular oxidative stress, endothelial injury, and thrombosis are intertwined processes that display a synergistic pathological effect in many cardiovascular diseases. Antithrombotic therapy with anticoagulant and/or antiplatelet agents, combined with interventions against vascular oxidative stress and/or inflammation, both boosting endothelial antithrombotic potential, could display a synergistic action in the treatment of thrombosis. Of the compounds 10a-h and 11a-d, shown to possess thrombin inhibitory activity, 11a-d were found to display radical scavenging activity, 10a, 10d, and 10f were demonstrated to inhibit lipid peroxidation of linoleic acid, and 10b and 10h inhibited soybean lipoxygenase. The observed combination of thrombin inhibition with lipid peroxidation and/or lipoxygenase inhibitory activity makes compounds 10 and 11 interesting candidates for further investigations towards multiple antithrombotic drugs.     

Lipofuscins and sclerotial differentiation in phytopathogenic fungi

Lipofuscins of lipidic and proteinaceous origin were identified by their excitation and emission spectra in phytopathogenic fungal representatives of different sclerotial differentiation types. Lipofuscin pigments in Sclerotium rolfsii, Rhizoctonia solani, Sclerotinia minor and Sclerotinia sclerotiorum showed similar excitation and emission maxima (ex-em 330–450, 330–450, 330–470 and 3307–470 nm, respectively). Sclerotial differentiation of these fungi was proceeded by a 4.2, 2.5, 2.7, 2.5 and 6, 2.9, 3.8, 3.1 fold increase of lipofuscin accumulation (per lipid and protein content), per respective fungus, as compared to their undifferentiated stage. Lipofuscin levels were higher in older than in younger mycelia and this phenomenon was more profound in S. rolfsii. Since lipofuscins are considered as indicators of oxidative stress, these data are in accordance with the hypothesis that suggests oxidative stress to be a common underlying factor in sclerotial differentiation of sclerotia-forming filamentous phytopathogenic fungi. This revised version was published online in June 2006 with corrections to the Cover Date.     

Is there any potential link among caspase-8, p-p38 MAPK and bcl-2 in clear cell renal cell carcinomas? A comparative immunohistochemical analysis with clinical connotations

Background

Management of endometrial precancerous lesions has been of much debate due to inconsistencies in their classification, natural history and histologic diagnosis. Endometrial hyperplasia constitutes a wide range of histomorphologic features associated with high intra and interobserver diagnostic variability. Although traditional microscopic diagnosis is by far the most applicable method and the gold standard for histomorphologic diagnosis, digitized image analysis has been used as a powerful adjunct to maximize the histologic data retrieval and to add some detailed objective criteria for correct diagnosis in difficult cases.

Methods

A series of 100 endometrial curettage specimens with diagnosis of endometrial hyperplasia or well differentiated adenocarcinoma were blindly reviewed by 5 pathologists; their intra and interobserver reproducibility determined and further compared to the objective morphometric data i.e. D-score and volume percent of stroma (VPS).

Results

The results were assessed using the weighted kappa statistics. Mean intraobserver kappa value was 0.8690 (99.44% agreement). Mean interobserver kappa values by diagnostic category were: simple hyperplasia without atypia: 0.7441; complex hyperplasia without atypia: 0.3379; atypical hyperplasia: 0.3473, and well-differentiated endometrioid carcinoma: 0.6428; with a kappa value of 0.5372 for all cases combined. Interobserver agreement was in substantial rate for simple hyperplasia (SH) and well differentiated adenocarcinoma (WDA) but was in fair limit for complex hyperplasia (CH) and atypical hyperplasia (AH). Intraobserver agreement was almost perfect. The specimens were divided in two groups according to the computerized morphometric analysis: Endometrial Hyperplasia (EH) ( D Score ≥ 1 or VPS ≥ 55%) and Endometrial Intraepithelial Neoplasia (EIN) (D-Score < 1 or VPS < 55%). Morphometric findings were closely compatible with routine WHO classification made by one expert pathologist; however; diagnosis of (CH) and (AH) made by other pathologists were not concordant with morphometric data.

Conclusion

It may be necessary to make some revisions in WHO classification for endometrial hyperplasia and precancerous lesions.     

Targeted and global pharmacometabolomics in everolimus-based immunosuppression: association of co-medication and lysophosphatidylcholines with dose requirement

Introduction

The immunosuppressive therapy with everolimus (ERL) after heart transplantation is characterized by a narrow therapeutic window and a substantial variability in dose requirement. Factors explaining this variability are largely unknown.

Objectives

Our aim was to evaluate factors affecting ERL metabolism and to identify novel metabolites associated with the individual ERL dose requirement to elucidate mechanisms underlying ERL dose response variability.

Method

We used liquid chromatography coupled with mass spectrometry for quantification of ERL metabolites in 41 heart transplant patients and evaluated the effect of clinical and genetic factors on ERL pharmacokinetics. Non-targeted plasma metabolic profiling by ultra-performance liquid chromatography and high resolution quadrupole-time-of-flight mass spectrometry was used to identify novel metabolites associated with ERL dose requirement.

Results

The determination of ERL metabolites revealed differences in metabolite patterns that were independent from clinical or genetic factors. Whereas higher ERL dose requirement was associated with co-administration of sodium-mycophenolic acid and the CYP3A5 expressor genotype, lower dose was required for patients receiving vitamin K antagonists. Global metabolic profiling revealed several novel metabolites associated with ERL dose requirement. One of them was identified as lysophosphatidylcholine (lysoPC) (16:0/0:0). Subsequent targeted analysis revealed that high levels of several lysoPCs were significantly associated with higher ERL dose requirement.

Conclusion

For the first time, this study describes distinct ERL metabolite patterns in heart transplant patients and detected potentially new drug–drug interactions. The global metabolic profiling facilitated the discovery of novel metabolites associated with ERL dose requirement that might represent new clinically valuable biomarkers to guide ERL therapy.

     

Neurohormonal and cytokine fluctuations following transcatheter closure for an atrial septal defect

Introduction

Inflammation and neurohormonal activation are considered to be involved in the development of earlier and/or later complications in congenital heart disease patients, even after a successful repair of the lesion. It is not yet clarified what is the role of the therapeutic interventions in the occurrence of such a response and how it could be associated with possible postoperative complications.

Aim

We sought to assess the inflammatory and neurohormonal response to transcatheter closure of secundum type atrial septal defects (ASD) over a six-month follow-up period. We also evaluated the association between the respective markers and catheterization data as well as echocardiographic measurements.

Methods

Plasma concentrations of interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), interleukin-10 (IL-10), N-terminal-proatrial natriuretic peptide (NT-proANP) and N-terminal-probrain natriuretic peptide (NT-proBNP) were assessed and echocardiographic measurements were performed in twenty-eight patients with atrial septal defect prior to, and at the first, second and sixth months post transcatheter closure. Thirty-three age-matched healthy volunteers were also enrolled.

Results

IL-6 plasma levels, although higher preoperatively, [physical logarithm (ln) IL-6: 3.37 ± 0.66 vs 2.92 ± 0.44 pg/ml, p = 0.015], reached control levels postoperatively, at the end of the third month, whereas TNF-α and IL-10 were not influenced by the procedure. NT-proANP levels were elevated preoperatively compared to the control group (ln NT-proANP 3.78 ± 0.572 vs 3.48 ± 0.30, p = 0.031), with a further significant increase during the 1st month (ln NT-proANP 3.78 ± 0.572 vs 4.2 ± 0.42, p = 0.006), following the pattern of the left atrial volume enlargement, and remained high even 6 months after the procedure .On the other hand, the initially normal concentrations of NT-proBNP, after a transient significant increase during the first month postoperatively (ln NT-proBNP 3.56 ± 0.94 vs 4.58 ± 0.91, p < 0.0001) returned to the controls’ levels at the end of the third month. Preoperative concentrations of NT-proANP positively correlated with NT-proBNP concentrations and pulmonary to systemic flow ratio (Qp/Qs).

Conclusions

Transcatheter closure could improve, on a mid- term basis, the inflammatory process but natriuretic peptides’ secretion continues in parallel with left atrial volume increase. Further follow up is required to determine the long-term progress of the inflammatory and neurohormonal response to the procedure.     

Mitochondrial consumption of cytosolic ATP: not so fast

In various pathologic circumstances depolarized mitochondria are thought to precipitate cell death by avidly consuming cytosolic ATP. However, for as long as the inner mitochondrial membrane remains intact the reversal potentials of the adenine nucleotide translocase (ANT) and that of F(0)-F(1) ATP synthase are strategically positioned so that they oppose import of cytosolic ATP into the matrix of respiration-impaired mitochondria. This arrangement also seems to protect against a hysteretic consumption of cytosolic ATP accumulating in the mitochondrial matrix, in view of the depolarization caused by inhibition of F(0)-F(1) ATP synthase by the endogenous protein IF1, yielding fast ANT reversal rates.     

Dimer/monomer status and in vivo function of salt‐bridge mutants of the plant UV‐B photoreceptor UVR8

UV RESISTANCE LOCUS8 (UVR8) is a photoreceptor for ultraviolet‐B (UV‐B) light that initiates photomorphogenic responses in plants. UV‐B photoreception causes rapid dissociation of dimeric UVR8 into monomers that interact with CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1) to initiate signal transduction. Experiments with purified UVR8 show that the dimer is maintained by salt‐bridge interactions between specific charged amino acids across the dimer interface. However, little is known about the importance of these charged amino acids in determining dimer/monomer status and UVR8 function in plants. Here we evaluate the use of different methods to examine dimer/monomer status of UVR8 and show that mutations of several salt‐bridge amino acids affect dimer/monomer status, interaction with COP1 and photoreceptor function of UVR8 in vivo. In particular, the salt‐bridges formed between arginine 286 and aspartates 96 and 107 are key to dimer formation. Mutation of arginine 286 to alanine impairs dimer formation, interaction with COP1 and function in vivo, whereas mutation to lysine gives a weakened dimer that is functional in vivo, indicating the importance of the positive charge of the arginine/lysine residue for dimer formation. Notably, a UVR8 mutant in which aspartates 96 and 107 are conservatively mutated to asparagine is strongly impaired in dimer formation but mediates UV‐B responses in vivo with a similar dose–response relationship to wild‐type. The UV‐B responsiveness of this mutant does not correlate with dimer formation and monomerisation, indicating that monomeric UVR8 has the potential for UV‐B photoreception, initiating signal transduction and responses in plants.