The G-protein–gated K+ channel,IKACh, is required for regulation of pacemaker activity and recovery of resting heart rate after sympathetic stimulation

Parasympathetic regulation of sinoatrial node (SAN) pacemaker activity modulates multiple ion channels to temper heart rate. The functional role of the G-protein–activated K⁺ current (I_KACh) in the control of SAN pacemaking and heart rate is not completely understood. We have investigated the functional consequences of loss of I_KACh in cholinergic regulation of pacemaker activity of SAN cells and in heart rate control under physiological situations mimicking the fight or flight response. We used knockout mice with loss of function of the Girk4 (Kir3.4) gene (Girk4^−/− mice), which codes for an integral subunit of the cardiac I_KACh channel. SAN pacemaker cells from Girk4^−/− mice completely lacked I_KACh. Loss of I_KACh strongly reduced cholinergic regulation of pacemaker activity of SAN cells and isolated intact hearts. Telemetric recordings of electrocardiograms of freely moving mice showed that heart rate measured over a 24-h recording period was moderately increased (10%) in Girk4^−/− animals. Although the relative extent of heart rate regulation of Girk4^−/− mice was similar to that of wild-type animals, recovery of resting heart rate after stress, physical exercise, or pharmacological β-adrenergic stimulation of SAN pacemaking was significantly delayed in Girk4^−/− animals. We conclude that I_KACh plays a critical role in the kinetics of heart rate recovery to resting levels after sympathetic stimulation or after direct β-adrenergic stimulation of pacemaker activity. Our study thus uncovers a novel role for I_KACh in SAN physiology and heart rate regulation.     

Tetraflagellochloris mauritanica gen. et sp. nov. (Chlorophyceae), a New Flagellated Alga from the Mauritanian Desert: Morphology,Ultrastructure, and Phylogenetic Framing

Morphological, ultrastructural, and molecular‐sequence data were used to assess the phylogenetic position of a tetraflagellate green alga isolated from soil samples of a saline dry basin near F'derick, Mauritania. This alga can grow as individual cells or form non‐coenobial colonies of up to 12 individuals. It has a parietal chloroplast with an embedded pyrenoid covered by a starch sheath and traversed by single parallel thylakoids, and an eyespot located in a parietal position opposite to the flagellar insertion. Lipid vacuoles are present in the cytoplasm. Microspectroscopy indicated the presence of chlorophylls a and b, with lutein as the major carotenoid in the chloroplast, while the eyespot spectrum has a shape typical of green‐algal eyespots. The cell has four flagella, two of them long and two considerably shorter. Sequence data from the 18S rRNA gene and ITS2 were obtained and compared with published sequences for green algae. Results from morphological and ultrastructural examinations and sequence analysis support the placement of this alga in the Chlorophyceae, as Tetraflagellochloris mauritanica L. Barsanti et A. Barsanti, gen. et sp. nov.     

Protein glutathionylation in health and disease

Background

It is now recognized that protein cysteines exist not only as free thiols or intramolecular disulfides, that help maintain the 3D structure of proteins, but can also undergo different types of oxidation, one of which is glutathionylation, or the formation of mixed disulfides with glutathione (GSH).

Scope of the review

We will discuss how proteins can undergo glutathionylation and how this can affect the protein characteristics/function. Glutathionylation is reversible and de-glutathionylation can be catalysed by protein thiol–disulfide oxidoreductases. Genetic modification of the expression of these enzymes, particularly glutaredoxin, using overexpression, knockout mice or siRNA, is becoming an important tool to study the role of protein glutathionylation. While in the past this post-translational modification was mainly known in the context of oxidative stress, measurement of glutathionylated proteins in patients is pointing out a potential importance if this modification in pathogenesis and could identify new biomarkers. We also wanted to point out the main findings in the role of glutathionylation in diseases and drug action.

Major conclusions

We identify two major open problems in the field, namely the complexity of the mechanisms responsible for glutathionylation and de-glutathionylation, as well as what makes a protein susceptible to glutathionylation.

General significance

This review underlines the peculiarities of this post-translational modification and their biological role. This article is part of a Special Issue entitled Cellular functions of glutathione.     

Immunomodulatory activity of SGI-110, a 5-aza-2′-deoxycytidine-containing demethylating dinucleotide

Purpose

Pharmacologic DNA hypomethylation holds strong promises in cancer immunotherapy due to its immunomodulatory activity on neoplastic cells. Searching for more efficient DNA hypomethylating agents to be utilized to design novel immunotherapeutic strategies in cancer, we investigated the immunomodulatory properties of the new DNA hypomethylating agent SGI-110, that is resistant to in vivo inactivation by cytidine deaminase.

Experimental design

Cutaneous melanoma, mesothelioma, renal cell carcinoma, and sarcoma cells were treated in vitro with SGI-110. RT-PCR, quantitative RT-PCR, quantitative methylation-specific PCR, and flow cytometric analyses were performed to investigate changes induced by SGI-110 in the constitutive immune profile of cancer cells. The recognition by gp100-specific CTL of gp100-positive melanoma cells, treated or not with SGI-110, was tested by LDH release assays.

Results

SGI-110 induced/up-regulated the expression of investigated cancer/testis antigens (CTA) (i.e., MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A10, GAGE 1-2, GAGE 1-6, NY-ESO-1, and SSX 1-5) in all cancer cell lines studied, both at mRNA and at protein levels. Quantitative methylation-specific PCR analyses identified a hypomethylation of MAGE-A1 and NY-ESO-1 promoters in SGI-110-treated neoplastic cells, demonstrating a direct role of pharmacologic DNA demethylation in CTA induction. SGI-110 also up-regulated the expression of HLA class I antigens and of ICAM-1, resulting in an improved recognition of cancer cells by gp100-specific CTL.

Conclusions

Our findings show that SGI-110 is a highly attractive therapeutic agent to comprehensively increase immunogenicity and immune recognition of neoplastic cells, and provide the scientific rationale for its clinical development to design novel chemo-immunotherapeutic approaches in cancer patients.     

8q12.1q12.3 de novo microdeletion involving the CHD7 gene in a patient without the major features of CHARGE syndrome: Case report and critical review of the literature

CHARGE syndrome is an autosomal dominant inherited disorder characterized by a specific and recognizable pattern of anomalies. De novo mutations or deletions of the gene encoding chromodomain helicase DNA binding protein 7 (CHD7) are the major cause of CHARGE syndrome. In this report, we describe a patient with a typical phenotype characterized by psychomotor retardation, hypertrichosis, facial asymmetry, synophria, failure to thrive, developmental delay and gastro-esophageal reflux, carrying a de novo 6.04 Mb interstitial deletion in 8q12.1q12.3 detected by single nucleotide polymorphism (SNP) array analysis. Despite the deletion includes CHD7 and although the patient shares some of the clinical features of the CHARGE syndrome, she does not fulfill the clinical criteria for this syndrome. To the best of our knowledge, this is the second case with an entire deletion of the CHD7 gene not leading to CHARGE syndrome and, for this reason, useful to expand and further delineate the clinical features associated with the 8q12.1q12.3 deletion. Furthermore, the literature review revealed that the phenotype secondary to duplications of the same region partially overlaps with the phenotype reported in this study. Selected genes that are present in the hemizygous state and which might be important for the phenotype of this patient, are discussed in context of the clinical features.     

The velvet complex governs mycotoxin production and virulence of Fusarium oxysporum on plant and mammalian hosts

Fungal pathogens provoke devastating losses in agricultural production, contaminate food with mycotoxins and give rise to life‐threatening infections in humans. The soil‐borne ascomycete Fusarium oxysporum attacks over 100 different crops and can cause systemic fusariosis in immunocompromised individuals. Here we functionally characterized VeA, VelB, VelC and LaeA, four components of the velvet protein complex which regulates fungal development and secondary metabolism. Deletion of veA, velB and to a minor extent velC caused a derepression of conidiation as well as alterations in the shape and size of microconidia. VeA and LaeA were required for full virulence of F. oxysporum on tomato plants and on immunodepressed mice. A critical contribution of velvet consists in promoting chromatin accessibility and expression of the biosynthetic gene cluster for beauvericin, a depsipeptide mycotoxin that functions as a virulence determinant. These results reveal a conserved role of the velvet complex during fungal infection on plants and mammals.     

Synthesis and cytotoxic activity evaluation of 2,3-thiazolidin-4-one derivatives on human breast cancer cell lines

It is well known that resveratrol (RSV) displayed cancer-preventing and anticancer properties but its clinical application is limited because of a low bioavailability and a rapid clearance from the circulation. Aim of this work was to synthesize pharmacologically active resveratrol analogs with an enhanced structural rigidity and bioavailability. In particular, we have synthesized a library of 2,3-thiazolidin-4-one derivatives in which a thiazolidinone nucleus connects two aromatic rings. Some of these compounds showed strong inhibitory effects on breast cancer cell growth. Our results indicate that some of thiazolidin-based resveratrol derivatives may become a new potent alternative tool for the treatment of human breast cancer.