CD36 Protein Influences Myocardial Ca2+ Homeostasis and Phospholipid Metabolism: CONDUCTION ANOMALIES IN CD36-DEFICIENT MICE DURING FASTING*

Sarcolemmal CD36 facilitates myocardial fatty acid (FA) uptake, which is markedly reduced in CD36-deficient rodents and humans. CD36 also mediates signal transduction events involving a number of cellular pathways. In taste cells and macrophages, CD36 signaling was recently shown to regulate store-responsive Ca²⁺ flux and activation of Ca²⁺-dependent phospholipases A₂ that cycle polyunsaturated FA into phospholipids. It is unknown whether CD36 deficiency influences myocardial Ca²⁺ handling and phospholipid metabolism, which could compromise the heart, typically during stresses. Myocardial function was examined in fed or fasted (18–22 h) CD36^−/− and WT mice. Echocardiography and telemetry identified conduction anomalies that were associated with the incidence of sudden death in fasted CD36^−/− mice. No anomalies or death occurred in WT mice during fasting. Optical imaging of perfused hearts from fasted CD36^−/− mice documented prolongation of Ca²⁺ transients. Consistent with this, knockdown of CD36 in cardiomyocytes delayed clearance of cytosolic Ca²⁺. Hearts of CD36^−/− mice (fed or fasted) had 3-fold higher SERCA2a and 40% lower phospholamban levels. Phospholamban phosphorylation by protein kinase A (PKA) was enhanced after fasting reflecting increased PKA activity and cAMP levels in CD36^−/− hearts. Abnormal Ca²⁺ homeostasis in the CD36^−/− myocardium associated with increased lysophospholipid content and a higher proportion of 22:6 FA in phospholipids suggests altered phospholipase A₂ activity and changes in membrane dynamics. The data support the role of CD36 in coordinating Ca²⁺ homeostasis and lipid metabolism and the importance of this role during myocardial adaptation to fasting. Potential relevance of the findings to CD36-deficient humans would need to be determined.     

High-level production and covalent immobilization of Providencia rettgeri penicillin G acylase (PAC) from recombinant Pichia pastoris for the development of a novel and stable biocatalyst of industrial applicability

A complete, integrated process for the production of an innovative formulation of penicillin G acylase from Providencia rettgeri(rPAC(P.rett))of industrial applicability is reported. In order to improve the yield of rPAC, the clone LN5.5, carrying four copies of pac gene integrated into the genome of Pichia pastoris, was constructed. The proteinase activity of the recombinant strain was reduced by knockout of the PEP4 gene encoding for proteinase A, resulting in an increased rPAC(P.rett) activity of approximately 40% (3.8 U/mL vs. 2.7 U/mL produced by LN5.5 in flask). A high cell density fermentation process was established with a 5-day methanol induction phase and a final PAC activity of up to 27 U/mL. A single step rPAC(P.rett) purification was also developed with an enzyme activity yield of approximately 95%. The novel features of the rPAC(P.rett) expressed in P.pastoris were fully exploited and emphasized through the covalent immobilization of rPAC(P.rett). The enzyme was immobilized on a series of structurally correlated methacrylic polymers, specifically designed and produced for optimizing rPAC(P.rett) performances in both hydrolytic and synthetic processes. Polymers presenting aminic functionalities were the most efficient, leading to formulations with higher activity and stability (half time stability >3 years and specific activity ranging from 237 to 477 U/g (dry) based on benzylpenicillin hydrolysis). The efficiency of the immobilized rPAC(P.rett) was finally evaluated by studying the kinetically controlled synthesis of beta-lactam antibiotics (cephalexin) and estimating the synthesis/hydrolysis ratio (S/H), which is a crucial parameter for the feasibility of the process.     

The RgpB C-terminal domain has a role in attachment of RgpB to the outer membrane and belongs to a novel C-terminal-domain family found in Porphyromonas gingivalis

Porphyromonas gingivalis produces outer membrane-attached proteins that include the virulence-associated proteinases RgpA and RgpB (Arg-gingipains) and Kgp (Lys-gingipain). We analyzed the P. gingivalis outer membrane proteome and identified numerous proteins with C-terminal domains similar in sequence to those of RgpB, RgpA, and Kgp, indicating that these domains may have a common function. Using RgpB as a model to investigate the role of the C-terminal domain, we expressed RgpB as a full-length zymogen (recombinant RgpB [rRgpB]), with a catalytic Cys244Ala mutation [rRgpB(C244A)], or with the C-terminal 72 amino acids deleted (rRgpB435) in an Arg-gingipain P. gingivalis mutant (YH522AB) and an Arg- and Lys-gingipain mutant (YH522KAB). rRgpB was catalytically active and located predominantly attached to the outer membrane of both background strains. rRgpB(C244A) was inactive and outer membrane attached, with a typical attachment profile for both background strains according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but in YH522KAB, the prodomain was not removed. Thus, in vivo, RgpB export and membrane attachment are independent of the proteolytic activity of RgpA, RgpB, or Kgp. However, for maturation involving proteolytic processing of RgpB, the proteolytic activity of RgpB, RgpA, or Kgp is required. The C-terminally-truncated rRgpB435 was not attached to the outer membrane and was located as largely inactive, discrete 71-kDa and 48-kDa isoforms in the culture supernatant and the periplasm. These results suggest that the C-terminal domain is essential for outer membrane attachment and may be involved in a coordinated process of export and attachment to the cell surface.     

Epigenetic status of the H19 locus in human oocytes following in vitro maturation

Imprinting is an epigenetic modification that is reprogrammed in the germ line and leads to the monoallelic expression of some genes. Imprinting involves DNA methylation. Maternal imprint is reset during oocyte growth and maturation. In vitro maturation (IVM) of oocytes may, therefore, interfere with imprint acquisition and/or maintenance. To evaluate if maturing human oocytes in vitro would be hazardous at the epigenetic level, we first determined the methylation profile of the H19 differentially methylated region (DMR). The methylation status of the H19 DMR seems particularly vulnerable to in vitro culture conditions. We analyzed oocytes at different stages of maturation following IVM, germinal vesicle (GV), metaphase I (MI), and metaphase II (MII), using the bisulfite mutagenesis technique. Our results indicated that the unmethylated specific maternal profile for the H19 DMR was stably established at the GV stage. The majority of MI-arrested oocytes exhibited an altered pattern of methylation, the CTCF-binding site being methylated in half of the DNA strands analyzed. Of the 20 MII oocytes analyzed, 15 showed the normal unmethylated maternal pattern, while 5 originating from two different patients exhibited a methylated pattern. These findings highlight the need for extended analysis on MII-rescued oocytes to appreciate the epigenetic safety of the IVM procedure, before it becomes a routine and practical assisted reproductive procedure.     

Distinct modulation of voltage-gated and ligand-gated Ca2+ currents by PPAR-γ agonists in cultured hippocampal neurons

Type 2 diabetes mellitus is a metabolic disorder characterized by hyperglycemia and is especially prevalent in the elderly. Because aging is a risk factor for type 2 diabetes mellitus, and insulin resistance may contribute to the pathogenesis of Alzheimer's disease (AD), anti-diabetic agents (thiazolidinediones-TZDs) are being studied for the treatment of cognitive decline associated with AD. These agents normalize insulin sensitivity in the periphery and can improve cognition and verbal memory in AD patients. Based on evidence that Ca²⁺ dysregulation is a pathogenic factor of brain aging/AD, we tested the hypothesis that TZDs could impact Ca²⁺ signaling/homeostasis in neurons. We assessed the effects of pioglitazone and rosiglitazone (TZDs) on two major sources of Ca²⁺ influx in primary hippocampal cultured neurons, voltage-gated Ca²⁺ channel (VGCC) and the NMDA receptor (NMDAR). VGCC- and NMDAR-mediated Ca²⁺ currents were recorded using patch-clamp techniques, and Ca²⁺ intracellular levels were monitored with Ca²⁺ imaging techniques. Rosiglitazone, but not pioglitazone reduced VGCC currents. In contrast, NMDAR-mediated currents were significantly reduced by pioglitazone but not rosiglitazone. These results show that TZDs modulate Ca²⁺-dependent pathways in the brain and have different inhibitory profiles on two major Ca²⁺ sources, potentially conferring neuroprotection to an area of the brain that is particularly vulnerable to the effects of aging and/or AD.     

The CYPome (Cytochrome P450 complement) of Aspergillus nidulans

The cytochromes P450 (CYPs) are found in all biological kingdoms and genome sequencing projects continue to reveal an ever increasing number. The principle aim of this paper is to identify the complete CYPome of Aspergillus nidulans from the genome sequence version AN.3 deposited at the Broad institute, assign the appropriate CYP nomenclature and define function where possible. The completed analysis revealed a total of 111 CYP genes, 3 of which were previously unknown and 8 pseudogenes, representing 89CYP families, 21 of which are unique. We have identified 28 potential gene clusters associated with one or more CYP genes and discussed those with putative PKS and NRPS associated function. The chromosomal location of the genes, predicted cellular location of the proteins and possible function(s) are discussed.     

Age-dependent oxidative stress: toward an irreversible failure in endothelial maintenance

In order to maintain cellular homeostasis against endogenous and exogenous aggressions, different cellular mechanisms of defence, maintenance and repair are continuously activated throughout life. Hormesis, a concept based on the fact that mild stresses protect cells against subsequent stresses, amplifies the efficacy of the cellular mechanisms of defence and repair. Ageing, senescence and ultimately death, result from the exhaustion of these mechanisms maintaining cellular functions. One of the major sources of vascular endothelial damage is oxidative stress. The age-dependent shift in the redox environment towards pro-oxidation contributes to a progressive compensatory remodelling of the endothelium, an accumulation of damages, and its dysfunction, the premises for atherosclerosis. We propose that in agreement with the concept of hormesis, a moderate exposure during endothelial maturation to mild physiological oxidative stressors determines -vascular longevity.     

Characterization of a novel MK3 splice variant from murine ventricular myocardium

p38 MAP kinase (MAPK) isoforms α, β, and γ, are expressed in the heart. p38α appears pro-apoptotic whereas p38β is pro-hypertrophic. The mechanisms mediating these divergent effects are unknown; hence elucidating the downstream signaling of p38 should further our understanding. Downstream effectors include MAPK-activated protein kinase (MK)-3, which is expressed in many tissues including skeletal muscles and heart. We cloned full-length MK3 (MK3.1, 384 aa) and a novel splice variant (MK3.2, 266 aa) from murine heart. For MK3.2, skipping of exons 8 and 9 resulted in a frame-shift in translation of the first 85 base pairs of exon 10 followed by an in-frame stop codon. Of 3 putative phosphorylation sites for p38 MAPK, only Thr-203 remained functional in MK3.2. In addition, MK3.2 lacked nuclear localization and export signals. Quantitative real-time PCR confirmed the presence of these mRNA species in heart and skeletal muscle; however, the relative abundance of MK3.2 differed. Furthermore, whereas total MK3 mRNA was increased, the relative abundance of MK3.2 mRNA decreased in MK2^?/? mice. Immunoblotting revealed 2 bands of MK3 immunoreactivity in ventricular lysates. Ectopically expressed MK3.1 localized to the nucleus whereas MK3.2 was distributed throughout the cell; however, whereas MK3.1 translocated to the cytoplasm in response to osmotic stress, MK3.2 was degraded. The p38α/β inhibitor SB203580 prevented the degradation of MK3.2. Furthermore, replacing Thr-203 with alanine prevented the loss of MK3.2 following osmotic stress, as did pretreatment with the proteosome inhibitor MG132. In vitro, GST-MK3.1 was strongly phosphorylated by p38α and p38β, but a poor substrate for p38δ and p38γ. GST-MK3.2 was poorly phosphorylated by p38α and p38β and not phosphorylated by p38δ and p38γ. Hence, differential regulation of MKs may, in part, explain diverse downstream effects mediated by p38 signaling.     

Pediatric idiopathic intracranial hypertension: clinical and demographic features

Idiopathic intracranial hypertension (IIH) is a clinical condition characterized by elevated intracranial pressure and absence of clinical, laboratory or radiographic evidence of central nervous system infection, vascular malformation, intracranial space occupying lesion or hydrocephalus. In the last years the raising understanding of pediatric IIH, especially concerning its demographics and epidemiology, has brought up to a redefinition of diagnostic criteria and reevaluation of pathogenesis and treatment. The authors reviewed the records of nineteen consecutive children with newly diagnosed IIH in order to compare demographic characteristics, clinical pictures and ophthalmologic aspects as optic disc evaluation and visual field evaluation, as well as treatment modalities and follow up. Beside obesity and female gender, potential alternative risk factors remains to be investigated, which need a good collaboration between neuro-ophthalmologists and pediatric neurologists.