TNP Analogues Inhibit the Virulence Promoting IP3-4 Kinase Arg1 in the Fungal Pathogen Cryptococcus neoformans.

New antifungals with unique modes of action are urgently needed to treat the increasing global burden of invasive fungal infections. The fungal inositol polyphosphate kinase (IPK) pathway, comprised of IPKs that convert IP3 to IP8, provides a promising new target due to its impact on multiple, critical cellular functions and, unlike in mammalian cells, its lack of redundancy. Nearly all IPKs in the fungal pathway are essential for virulence, with IP3-4 kinase (IP3-4K) the most critical. The dibenzylaminopurine compound, N2-(m-trifluorobenzylamino)-N6-(p-nitrobenzylamino)purine (TNP), is a commercially available inhibitor of mammalian IPKs. The ability of TNP to be adapted as an inhibitor of fungal IP3-4K has not been investigated. We purified IP3-4K from the human pathogens, Cryptococcus neoformans and Candida albicans, and optimised enzyme and surface plasmon resonance (SPR) assays to determine the half inhibitory concentration (IC50) and binding affinity (KD), respectively, of TNP and 38 analogues. A novel chemical route was developed to efficiently prepare TNP analogues. TNP and its analogues demonstrated inhibition of recombinant IP3-4K from C. neoformans (CnArg1) at low µM IC50s, but not IP3-4K from C. albicans (CaIpk2) and many analogues exhibited selectivity for CnArg1 over the human equivalent, HsIPMK. Our results provide a foundation for improving potency and selectivity of the TNP series for fungal IP3-4K.

A Pathogenic Galactosidase A Mutation Coexisting With an MYBPC3 Mutation in a Female Patient With Hypertrophic Cardiomyopathy.

The coexistence of GLA (Pro259Ser, c.775C>T) and MYBPC3 (c.1351+2T>C) mutations was found in a female patient with hypertrophic cardiomyopathy. Histology documented abundant vacuolisation with osmiophilic lamellar bodies and positive Gb3 immunohistochemistry. In the presence of a hypertrophic cardiomyopathy phenotype, the systematic search for unusual findings is mandatory to rule out a phenocopy.

The MTA1 subunit of the nucleosome remodeling and deacetylase complex can recruit two copies of RBBP4/7.

The nucleosome remodeling and deacetylase (NuRD) complex remodels the genome in the context of both gene transcription and DNA damage repair. It is essential for normal development and is distributed across multiple tissues in organisms ranging from mammals to nematode worms. In common with other chromatin-remodeling complexes, however, its molecular mechanism of action is not well understood and only limited structural information is available to show how the complex is assembled. As a step towards understanding the structure of the NuRD complex, we have characterized the interaction between two subunits: the metastasis associated protein MTA1 and the histone-binding protein RBBP4. We show that MTA1 can bind to two molecules of RBBP4 and present negative stain electron microscopy and chemical crosslinking data that allow us to build a low-resolution model of an MTA1-(RBBP4)2 subcomplex. These data build on our understanding of NuRD complex structure and move us closer towards an understanding of the biochemical basis for the activity of this complex.

Differential atrial versus ventricular ANKRD1 gene expression is oppositely regulated at diastolic heart failure.

Diastolic heart failure (DHF) was produced in 6-day-old piglets by intravenous administration of Doxorubicin, and ANKRD1 protein and mRNA levels were determined in atrial (A) and ventricular (V) chambers of failing vs control hearts. In controls, ANKRD1 showed a left-right (L-R) asymmetric distribution with protein levels 2-fold higher in the LA as compared to the RA, and 8-fold higher in the LV than the RV. In failing hearts, ANKRD1 levels were augmented about 2-fold in each ventricle but equally reduced in both atria as compared to controls. ANKRD1 downregulation in atria is discussed as a process associated with advanced DHF.

Esterase-like and fibronectin-like polypeptides share similar sex-cell-biased patterns in the gonad of hermaphroditic and gonochoric species of bivalve mollusks.

The mechanism of sexualization of the tubular gonad in seawater bivalves is unknown, and no information regarding the genes involved in this process is yet available, except for the identification of esterase (Est)-like "male-associated polypeptide" in the male gonad of Mytilus galloprovincialis. Our present work reveals distinct protein profiles specific for the testicular or ovarian portion of the ovotestis of Pecten maximus. Two proteins exhibiting testis- or ovary-dependent enrichment in the ovotestis have been identified and partially characterized as Est-like and fibronectin (Fn)-like polypeptides, respectively. Immunofluorescence has demonstrated a close association between the localization of these polypeptides and the gonad tubule network and interstitial stroma of the ovotestis of P. maximus. We also present evidence of Est-like and Fn-like protein enrichment, respectively, in testicular and ovarian tissue in hermaphroditic, sex-reversal, and gonochoric species of seawater bivalves. Together, the results (1) strongly suggest that sex-cell-biased expression of Est-like and Fn-like polypeptides in gonad tissue is a widespread phenomenon among bivalve mollusks, despite the high diversification of their sexual patterns, (2) confirm and expand our previous demonstration of sex-biased protein expression in M. galloprovincialis, and (3) indicate a direct link between germ cell differentiation and sexual specialization of the bivalve somatic gonad.

Pdlim2, a novel PDZ-LIM domain protein, interacts with alpha-actinins and filamin A.

PURPOSE: To characterize properties of Pdlim2, a novel PDZ and LIM domain-containing protein. METHODS: cDNA encoding Pdlim2 was identified in a cDNA library of transcripts expressed in the tissues of the rat eye irido-corneal angle. The expression pattern of the Pdlim2 gene was studied by Northern blot analysis and in situ hybridization. Proteins interacting with Pdlim2 were identified by pull-down assay and mass spectrometry. Intracellular localization of Pdlim2 was investigated by confocal microscopy. RESULTS: Rat Pdlim2 protein belongs to the ALP subfamily of proteins containing the PDZ domain in the N-terminal portion and the LIM domain in the C-terminal portion of the protein. The Pdlim2 gene was specifically expressed in the corneal epithelial cells, but not in the corneal stroma and endothelium nor in other ocular tissues. Pdlim2 was also expressed in the lung. In rat corneal and lung extracts, alpha-actinin-1, alpha-actinin-4, filamin A, and myosin heavy polypeptide 9 were co-immunoprecipitated with Pdlim2. Myosin VI was co-immunoprecipitated with Pdlim2 from corneal but not lung extracts. alpha-Actinins were the most abundant among immunoprecipitated proteins. Direct interaction of Pdlim2 with alpha-actinins and filamin was confirmed using pull-down assays and gel overlay assay with purified proteins. Pdlim2 and alpha-actinins were co-localized mainly to stress fibers after transfection into COS-7 cells. In transfected COS-7 cells, complexes of Pdlim2 and alpha-actinin-1 were preferentially located along the basal aspect. CONCLUSIONS: These results suggest that Pdlim2, like other ALP subfamily members, may act as an adapter that directs other proteins to the cytoskeleton.

Left-right asymmetric ventricular expression of CARP in the piglet heart: regional response to experimental heart failure.

BACKGROUND AND AIM: Cardiac ankyrin repeat protein (CARP), whose expression is down-regulated in response to doxorubicin (Dox) in vitro, has been proposed to be a marker of experimentally-induced cardiac hypertrophy in rodent models. In piglets, the rapid hypertrophy rate of the left ventricle (LV) as compared to that of the right ventricle (RV) represents a natural model of asymmetric ventricular enlargement. We tested whether CARP expression correlates with postnatal ventricular hypertrophy and to what extent CARP can be sensitive to Dox treatment in vivo. METHODS: CARP mRNA and protein levels were quantified (by Northern blot hybridization, semi-quantitative RT-PCR and Western blot) in the piglet heart, both during early postnatal development and upon Dox-induced cardiomyopathy (Dox-CM). RESULTS: The study revealed: (1) significantly augmented CARP mRNA and protein levels in the LV compared to the RV resulting in left vs. right asymmetry in ventricular CARP expression throughout early postnatal development; (2) dose- and chamber-dependent CARP mRNA and protein enrichment in ventricular myocardium in response to Dox; and (3) abolishment of asymmetric patterns of ventricular CARP expression at heart failure resulting from Dox-CM. CONCLUSIONS: (1) CARP is differentially regulated in the LV and RV during both postnatal development and disease; and (2) monitoring of ventricular CARP expression patterns can be used for further analysis of transition from compensated to overt heart failure.

Mussel MAP, a major gonad-duct esterase-like protein, is released into sea water as a dual constituent of the seminal fluid and the spermatozoon.

Our interest in the comparative analysis of male reproductive-tract esterases in different animal groups has led us to undertake a detailed study of the Mytilus galloprovincialis male-associated polypeptide (MAP) throughout the mussel gonad-duct tract and at spawning. The results of this work indicate that MAP is a major protein in M. galloprovincialis semen, with dual presence in both sperm cells and cell-free seminal fluid. Shortly after spawning, the released sperm mass is subdivided in diffused cloudy-like and thread-shaped 'clots', in which a soluble-phase MAP may persist as long as the clots keep their compact form. Additional experiments involving the incubation of spawned spermatozoa at increasing Triton X-100 concentrations demonstrated that MAP is also strongly associated with sperm cells. These results were further validated by immunofluorescent staining, which revealed that MAP is localized in the mid-piece region of spawned spermatozoa. This unexpected finding raises the possibility that MAP may play a role in sperm fertility in bivalves. Using whole-mount histology and micromanipulation techniques, we studied the structural patterning of the mantle gonad-duct network and assessed the sampling of luminal contents from the ducts. Of particular interest is the observation that MAP content in the luminal fluid increases from the lumen of the spermatogenic tubules to that of the collecting gonad ducts, where MAP is detected at a very high concentration. These high levels may lead to a significant presence of MAP in semen and consequently to a prolonged survival of sperm spawned at sea. In addition, data related to the potential structural similarity between mussel MAP and esterase S of the Drosophila virilis ejaculatory bulb are presented and discussed. Finally, we show that the 64kDa protein of human semen reveals positive cross-reactivity with antibodies directed against Mytilus MAP and Drosophila esterase S. Taken together, the results reveal mussel MAP as the only esterase-like protein described so far whose distribution in the gonad and semen can be specifically associated with maturation, transport, emission and survival of spermatozoa outside.