Cytotoxic chalcones and flavanones from the tree bark of Cryptocarya costata

A new flavanone, 7-hydroxy-5,6-dimethoxyflavanone (1), together with three other flavonoids, didymocarpin (2), 2',4'-dihydroxy-5',6'-dimethoxychalcone (3), and isodidymocarpin (4), had been isolated from the methanol extract of the tree bark of Cryptocarya costata. The structures of these compounds were determined based on spectral evidence, including UV, IR, 1-D and 2-D NMR, and mass spectra. Cytotoxic properties of compounds 1-4 were evaluated against murine leukemia P-388 cells. The chalcones 3 and 4 were found to have substantial cytotoxicity with IC50 of 5.7 and 11.1 microM, respectively.     

Docosahexaenoic acid attenuates microglial activation and delays early retinal degeneration

Microgliosis is a common phenomenon in neurodegenerative disorders including retinal dystrophies. We performed a detailed characterization of activated microglia in the retinoschisin (Rs1h)-deficient (Rs1h^−/Y) mouse model of inherited retinal degeneration. To visualize and isolate microglia, we crossed Rs1h^−/Y animals with transgenic MacGreen mice, which express green fluorescent protein under the control of the macrophage-specific csf1r promoter. Activated microglia were detected in retinal sections and whole-mounts of early postnatal MacGreen/Rs1h^−/Y mice before the onset of overt neuronal cell death. These activated microglia contained prominent lipid droplets and analysis of the retinal lipid composition showed decreased docosahexaenoic acid (DHA) levels in Rs1h^−/Y retinas. To establish a link between microglia activation, reduced DHA levels, and neurodegeneration, a dietary intervention study was performed. Female Rs1h^−/− mice and their Rs1h^−/Y litter were either subjected to a diet enriched with DHA, or a control chow lacking DHA. Supplementation with DHA enhanced photoreceptor survival and converted activated microglia to a quiescent phenotype. Furthermore, DHA, but not docosapentaenoic acid or adrenic acid reduced pro-inflammatory gene expression, migration, and lipid accumulation of cultured BV-2 microglia. We conclude that retinal DHA levels control the activity of microglia and thereby may affect the progression and extent of retinal degeneration.     

Spermatogenesis in Boccardiella hamata (Polychaeta: Spionidae) from the Sea of Japan: sperm formation mechanisms as characteristics for future taxonomic revision

Reunov, A.A., Yurchenko, O.V., Alexandrova, Y.N. and Radashevsky, V.I. 2009. Spermatogenesis in Boccardiella hamata (Polychaeta: Spionidae) from the Sea of Japan: sperm formation mechanisms as characteristics for future taxonomic revision. —Acta Zoologica (Stockholm) 91 : 477–456. To characterize novel features that will be useful in the discussion and validation of the spionid polychaete Boccardiella hamata from the Sea of Japan, the successive stages of spermatogenesis were described and illustrated. Spermatogonia, spermatocytes and early spermatids are aflagellar cells that develop synchronously in clusters united by a cytophore. At the middle spermatid stage, the clusters undergo disintegration and spermatids produce flagella and float separately in coelomic fluid as they transform into sperm. Spermatozoa are filiform. The ring‐shaped storage platelets are located along the anterior nuclear area. The nucleus is cupped by a conical acrosome. A nuclear plate is present between the acrosome and nucleus. The nucleus is a cylinder with the implantation fossa throughout its length and with the anterior part of the flagellum inside the fossa. There is only one centriole, serving as a basal body of the flagellum, situated in close vicinity of the acrosomal area. A collar of four mitochondria is located under the nuclear base. The ultrastructure of B. hamata spermatozoa from the Sea of Japan appears to be close to that of B. hamata from Florida described by Rice (Microscopic Anatomy of Invertebrates, Wiley‐Liss, Inc., New York, 1992), suggesting species identity of the samples from the two regions. However, more detailed study of Florida’s B. hamata sperm is required for a reliable conclusion concerning the similarity of these two polychaetes. In addition to sperm structure, features such as the cytophore‐assigned pattern of spermatogenic cell development, the synchronous pattern of cell divisions, the non‐flagellate early spermatogenic stages, and the vesicle amalgamation that drives meiotic cell cytokinesis and spermatid diorthosis will likely be useful in future testing of the validity of B. hamata and sibling species throughout the world.     

The Aspergillus fumigatus cspA Gene Encoding a Repeat-Rich Cell Wall Protein Is Important for Normal Conidial Cell Wall Architecture and Interaction with Host Cells

cspA (for cell surface protein A) encodes a repeat-rich glycophosphatidylinositol (GPI)-anchored cell wall protein (CWP) in the pathogenic fungus Aspergillus fumigatus. The number of repeats in cspA varies among isolates, and this trait is used for typing closely related strains of A. fumigatus. We have previously shown that deletion of cspA is associated with rapid conidial germination and reduced adhesion of dormant conidia. Here we show that cspA can be extracted with hydrofluoric acid (HF) from the cell wall, suggesting that it is a GPI-anchored CWP. The cspA-encoded CWP is unmasked during conidial germination and is surface expressed during hyphal growth. Deletion of cspA results in weakening of the conidial cell wall, whereas its overexpression increases conidial resistance to cell wall-degrading enzymes and inhibits conidial germination. Double mutant analysis indicates that cspA functionally interacts with the cell wall protein-encoding genes ECM33 and GEL2. Deletion of cspA together with ECM33 or GEL2 results in strongly reduced conidial adhesion, increased disorganization of the conidial cell wall, and exposure of the underlying layers of chitin and β-glucan. This is correlated with increasing susceptibility of the ΔcspA, ΔECM33, and ΔcspA ΔECM33 mutants to conidial phagocytosis and killing by human macrophages and hyphal damage induced by neutrophils. However, these strains did not exhibit altered virulence in mice with infected lungs. Collectively, these results suggest a role for cspA in maintaining the strength and integrity of the cell wall.The saprophytic mold Aspergillus fumigatus is an emerging pathogen and the major causative agent of invasive aspergillosis, a life-threatening disease primarily affecting immunocompromised patients (12, 16, 38).Molecular analyses have revealed numerous virulence attributes that enable A. fumigatus to infect the human host, including the production of toxins, the ability to acquire nutrients and iron under limiting conditions, and the presence of protective mechanisms that degrade oxygen radicals released by the host immune cells (7).The fungal cell wall plays a crucial role in infection. In A. fumigatus, as in other pathogenic fungi, the cell wall protects the fungus and interacts directly with the host immune system. It is an elastic, dynamic, and highly regulated structure and is essential for growth, viability, and infection. The fungal cell wall is a unique structure and therefore a specific target for antifungal drugs. The cell wall of A. fumigatus is composed of a polysaccharide skeleton interlaced and coated with cell wall proteins (CWPs). The main building blocks of the polysaccharide skeleton are an interconnected network of glucan, chitin, and galactomannan polymers (26). The major class of fungal CWPs is the glycophosphatidylinositol (GPI)-modified proteins (8,–11, 14).We recently identified and characterized A. fumigatus CWPs containing tandem repeats (27). Repeats are hot spots of genetic change: because of replication slippage and recombination, repeats can undergo rapid changes in copy number, leading to natural variability among different isolates and allowing faster adaptation to new environments (23). In Saccharomyces cerevisiae, for example, an increase in the number of coding repeats in the FLO1 adhesin-encoding gene correlates with an increase in adhesion to the plastics used in medical devices (44,–46). Similarly, repeat variation in the Candida albicans ALS3 adhesin changes its cellular binding specificity (34). Moreover, clinical C. albicans isolates show variability in the number of repeats in various cell surface genes, suggesting that this recombination process could play a role during infection, allowing cells to adapt rapidly to a fluctuating environment and/or evade the host immune system (34, 49, 50).We identified four genes encoding putative A. fumigatus GPI-anchored CWPs (AFUA_3G08990 [termed cspA for cell-surface protein A [4], AFUA_2G05150 [MP-2], AFUA_4G09600, and AFUA_6G14090) containing variable numbers of repeats among patient isolates (27). In A. fumigatus WT strain AF 293, cspA encodes a 433-amino-acid-long protein containing a putative leader sequence and GPI modification site. cspA lacks recognizable catalytic domains, and homologous genes are found only in species of Aspergillus. Most interesting is that the gene encodes a 188-amino-acid-long serine-threonine-proline-rich N-terminal region followed by a large size-variable six-amino-acid serine-proline [P-G-Q-P-S-(A/V)]-rich tandem repeat region showing significant homology to the repeat domains found in mammalian type XXI collagen. The number of repeats varies between 18 and 47 (24 to 65% of the length of the protein) in different isolates of A. fumigatus. The strains used in this study, AF 293 and CBS 144.89, contain 32 and 28 repeats, respectively.Deletion of cspA resulted in a phenotype characterized by rapid conidial germination and reduced adhesion to extracellular matrix (ECM), which suggests that cspA participates in defining cell surface properties. Highlighting the importance of this gene, Balajee et al. (4) showed that variations in the cspA nucleotide repeat sequence can be used to type closely related pathogenic isolates of A. fumigatus and identify outbreak clusters occurring in hospitals (3, 4).In this work, we undertook a detailed study of cspA. We analyzed the expression pattern of the protein encoded by cspA and its attachment to the cell wall. We prepared and analyzed A. fumigatus mutant strains in which cspA was overexpressed or deleted in combination with additional cell wall-associated genes. Results indicate that the protein encoded by cspA is GPI anchored to the cell wall and is unmasked during conidial germination. cspA deletion weakens the cell wall and results in rapid conidial germination, whereas cspA overexpression increases conidial resistance to protoplasting and inhibits conidial germination. cspA functionally interacts with the genes ECM33 and GEL2, which encode cell wall-associated proteins, resulting primarily in profound defects in conidial cell wall organization. The cspA ECM33 double mutant exhibited greater susceptibility to killing by human macrophages and hyphal damage induced by neutrophils. The implications of our findings are discussed.     

Kinetics of antigen binding to antibody microspots: strong limitation by mass transport to the surface

It is well documented that diffusion has generally a strong effect on the binding kinetics in the microtiter plate immunoassays. However, a systematic quantitative experimental evaluation of the microspot kinetics is still missing in the literature. Our work aims at filling this important gap of knowledge on the example of antigen binding to antibody microspots. A mathematical model was derived within the framework of two-compartment model and applied to the quantitative analysis of the experimental data obtained for typical antibody microspot assays. A strong mass-transport dependence of the antigen-antibody microspot kinetics was identified to be one of the main restrictions of this new technology. The binding reactions are slowed down in the microspot immunoassays by several orders of magnitude as compared with the corresponding well-stirred bulk reactions. The task to relax the mass-transport limitations should thus be one of the most important issues in designing the antibody microarrays. These limitations notwithstanding, the detection range of more than five orders of magnitude and the high sensitivity in the low femtomolar range were experimentally achieved in our study, demonstrating thus an enormous potential of this highly capable technology.     

Absence of mechanical allodynia and Abeta-fiber sprouting after sciatic nerve injury in mice lacking membrane-type 5 matrix metalloproteinase

Matrix metalloproteinases (MMPs) are a family of endopeptidases that degrade extracellular matrix components. Membrane-type 5 MMP (MT5-MMP/MMP-24) was identified as neuron-specific, and is believed to contribute to neuronal circuit formation and plasticity. To elucidate its function in vivo, we have generated mice lacking MT5-MMP by gene targeting. MT5-MMP-deficient mice were born without obvious morphological abnormalities. No apparent histological defects were observed in the nervous system either. However, MT5-MMP-deficient mice did not develop neuropathic pain with mechanical allodynia after sciatic nerve injury, though responses to acute noxious stimuli were normal. Neuropathic pain induced by peripheral nerve lesions is known to accompany structural reorganization of the nervous system. Intraneural injection of cholera toxin B subunit, a transganglionic tracer, into the injured sciatic nerve of wild-type mice revealed that the myelinated Abeta-fiber primary afferents sprouted from laminae III-VI of the dorsal horn of the spinal cord and invaded lamina II. However, no such sprouting and invasion of Abeta-fibers were observed in MT5-MMP-deficient mice. These findings suggest that MT5-MMP is essential for the development of mechanical allodynia and plays an important role in neuronal plasticity in this mouse model.     

Lipids of the zygomycete Absidia corymbifera F-965

The cell lipids of the zygomycete Absidia corymbifera F-965 extracted with isopropanol and CHCl3-MeOH mixtures at the exponential growth phase comprise 20+/-2% of mycelium dry wt. The lipids consist of: triacylglycerols (51% of the total lipids extracted), diacylglycerols (9%), monoacylglycerols (3%), ergosterol (5%), ergosterol peroxide (5alpha,8alpha-epidioxyergosta-6,22-diene-3beta-ol) (3%), fatty-acid esters of ergosterol (less than 0.5%), free fatty acids (4%), glucocerebroside (3%), and glycerophospholipids (22%). The main phospholipids are phosphatidylethanolamine (39% of the total phospholipids), phosphatidyl-myo-inositol (17%), diphosphatidylglycerol (12%), phosphatidic acid (7%), phosphatidylcholine (6%), phosphatidylglycerol (3%), and two unusual phospholipids reported earlier, N-acetylphosphatidylethanolamine (7%) and N-ethoxycarbonyl phosphatidylethanolamine (9%). In addition, two unknown acidic phospholipids are present in traces. Saturated fatty acids of the lipids are dominated by n-hexadecanoic acid and unsaturated ones by octadecenoic acid; octadecadienoic and octadecatrienoic acids are present in lesser amounts. Ergosterol peroxide as well as the above glucocerebroside which contains 9-methylsphinga-4(E),9(E)-dienine have first been revealed in zygomycetes.     

Clusterin,an abundant serum factor,is a possible negative regulator of MT6-MMP/MMP-25 produced by neutrophils

MT6-MMP/MMP-25 is the latest member of the membrane-type matrix metalloproteinase (MT-MMP) subgroup in the MMP family and is expressed in neutrophils and some brain tumors. The proteolytic activity of MT6-MMP has been studied using recombinant catalytic fragments and shown to degrade several components of the extracellular matrix. However, the activity is possibly modulated further by the C-terminal hemopexin-like domain, because some MMPs are known to interact with other proteins through this domain. To explore the possible function of this domain, we purified a recombinant MT6-MMP with the hemopexin-like domain as a soluble form using a Madin-Darby canine kidney cell line as a producer. Mature and soluble MT6-MMP processed at the furin motif was purified as a 45-kDa protein together with a 46-kDa protein having a single cleavage in the hemopexin-like domain. Interestingly, 73- and 70-kDa proteins were co-purified with the soluble MT6-MMP by forming stable complexes. They were identified as clusterin, a major component of serum, by N-terminal amino acid sequencing. MT1-MMP that also has a hemopexin-like domain did not form a complex with clusterin. MT6-MMP forming a complex with clusterin was detected in human neutrophils as well. The enzyme activity of the soluble MT6-MMP was inactive in the clusterin complex. Purified clusterin was inhibitory against the activity of soluble MT6-MMP. On the other hand, it had no effect on the activities of MMP-2 and soluble MT1-MMP. Because clusterin is an abundant protein in the body fluid in tissues, it may act as a negative regulator of MT6-MMP in vivo.     

Membrane-type 5 matrix metalloproteinase is expressed in differentiated neurons and regulates axonal growth.

Expression of membrane-type (MT) 5 matrix metalloproteinase (MMP) in the mouse brain was examined. MT5-MMP was expressed in the cerebrum in embryos, but it declined after birth. In contrast, expression in the cerebellum started to increase postnatally and continued thereafter. The cells expressing MT5-MMP were postmitotic neurons that showed gelatinolytic activities. Specific expression of MT5-MMP was observed in the neurons but not in the glial cells when embryonal mouse carcinoma P19 cells were differentiated in vitro by retinoic acid treatment. Neurons isolated from dorsal root ganglia also expressed MT5-MMP, and it was localized at the edge of growth cone. Proteoglycans inhibit neurite extension and regulate synaptogenesis. The inhibitory effect of the proteoglycans on neurite extension of dorsal root ganglia neurons was effectively eliminated by recombinant MT5-MMP. Thus, MT5-MMP expressed in neurons may play a role in axonal growth that contributes to the regulation of neural network formation.