Differential involvement of rat sperm choline glycerophospholipids and sphingomyelin in capacitation and the acrosomal reaction

Rat spermatozoa main lipid classes and their fatty acids were studied to assess their possible changes in capacitation and the acrosomal reaction (AR), induced in vitro. Capacitation-associated protein tyrosine phosphorylation, and the efflux of 30% of the total cholesterol from gametes to the medium, took place concomitantly with the release of a similar percentage, i.e., a larger amount, of the total phospholipid, mostly after hydrolysis of the major choline glycerophospholipids (CGP). Main medium lipid metabolites after capacitation were lyso-CGP and polyenoic fatty acids typical of CGP (22:4n-9, 22:5n-6), as free fatty acids (FFA). The AR, induced by a calcium ionophore, resulted in further phospholipid loss, but the produced metabolites remained in the gametes. CGP decrease in AR accounted for some additional FFA and lyso-CGP, but mostly for (22:5n-6-rich) diglycerides. Hydrolysis of sphingomyelins (SM) to ceramides also occurred, mostly affecting species with very long chain polyenoic fatty acids. Quantitatively, CGP and SM were the lipid classes decreasing the most after capacitation and AR, respectively. The massive cholesterol and phospholipid loss from the gametes during capacitation is thus associated with protein phosphorylation, a function that has been located to the sperm tail. The lipid metabolites produced during AR, by accumulating in the gamete heads, could be implicated in sperm–oocyte interactions.     

Homocysteine Induces Hypophosphorylation of Intermediate Filaments and Reorganization of Actin Cytoskeleton in C6 Glioma Cells

In this study, we investigated the actions of high homocysteine (Hcy) levels (100 and 500 μM) on the cytoskeleton of C6 glioma cells. Results showed that the predominant cytoskeletal response was massive formation of actin-containing filopodia at the cell surface that could be related with Cdc42 activation and increased vinculin immunocontent. In cells treated with 100 μM Hcy, folic acid, trolox, and ascorbic acid, totally prevented filopodia formation, while filopodia induced by 500 μM Hcy were prevented by ascorbic acid and attenuated by folic acid and trolox. Moreover, competitive NMDA ionotropic antagonist DL-AP5 totally prevented the formation of filopodia in both 100 and 500 μM Hcy treated cells, while the metabotropic non-selective group I/II antagonist MCPG prevented the effect of 100 μM Hcy but only slightly attenuated the effect induced by of 500 μM Hcy on actin cytoskeleton. The competitive non-NMDA ionotropic antagonist CNQX was not able to prevent the effects of Hcy on the reorganization of actin cytoskeleton in the two concentrations used. Also, Hcy-induced hypophosphorylation of vimentin and glial fibrillary acidic protein (GFAP) and this effect was prevented by DL-AP5, MCPG, and CNQX. In conclusion, our results show that Hcy target the cytoskeleton of C6 cells probably by excitoxicity and/or oxidative stress mechanisms. Therefore, we could propose that the dynamic restructuring of the actin cytoskeleton of glial cells might contribute to the response to the injury provoked by elevated Hcy levels in brain.     

Amino acid based enantiomerically pure 3-substituted benzofused heterocycles: A new class of antithrombotic agents

A diverse group of novel medium ring heterocycles derived from naturally abundant proteinogenic amino acids were evaluated for their potency towards antithrombotic activity. The more potent benzofused oxazepine and oxazocine scaffolds were diversified by incorporating different amino acids at the position number 3. Further the effect of ring size has also been taken into account and it was observed that the eight-membered oxazocines ane more potent compared to the corresponding oxazepines.     

Disulfide cross-linking reveals a site of stable interaction between C-terminal regulatory domains of the two MalK subunits in the maltose transport complex

Understanding the structure and function of the ATP-binding cassette (ABC) transporters is very important because defects in ABC transporters lie at the root of several serious diseases including cystic fibrosis. MalK, the ATP-binding cassette of the maltose transporter of Escherichia coli, is distinct from most other ATP-binding cassettes in that it contains an additional C-terminal regulatory domain. The published structure of a MalK dimer is elongated with C-terminal domains at opposite poles (Diederichs, K., Diez, J., Greller, G., Muller, C., Breed, J., Schnell, C., Vonrhein, C., Boos, W., and Welte, W. (2000) EMBO J. 19, 5951-5961). Some uncertainty exists as to whether the orientation of MalK in the dimer structure is correct. Superpositioning of the N-terminal domains of MalK onto the ATP-binding domains of an alternate ABC dimer, in which ATP is bound along the dimer interface between Walker A and LSGGQ motifs, places both N- and C-terminal domains of MalK along the dimer interface. Consistent with this model, a cysteine substitution at position 313 in the C-terminal domain of an otherwise cysteine-free MalK triggered disulfide bond formation between two MalK subunits in an intact maltose transporter. Disulfide bond formation did not inhibit the function of the transporter, suggesting that the C-terminal domains of MalK remain in close proximity throughout the transport cycle. Enzyme IIAglc still inhibited the ATPase activity of the disulfide-linked transporter indicating that the mechanism of inducer exclusion was unaffected. These data support a model for ATP hydrolysis in which the C-terminal domains of MalK remain in contact whereas the N-terminal domains of MalK open and close to allow nucleotide binding and dissociation.     

Aromatic-aromatic interactions in and around alpha-helices

To understand the role of aromatic-aromatic interactions in imparting specificity to the folding process, the geometries of four aromatic residues with different sequence spacing, located in alpha-helices or five residues from helical ends, interacting with each other have been elucidated. The geometry is found to depend on the sequence difference. Specific interactions (C-H...pi and N-H...pi) which result from this geometry may cause a given pair of residues (such as Phe-His) with a particular sequence difference to occur more than expected. The most conspicuous residue in an aromatic pair in the context of helix stability is His, which is found at the last (C1) position or the two positions (Ncap and Ccap) immediately flanking the helix. An alpha-helix and a contiguous 3(10)-helix or two helices separated by a non-helical residue can have interacting aromatic pairs, the geometry of interaction and the relative orientation between the helices being rather fixed. Short helices can also have interacting residues from either side.     

Segregational and structural instability of recombinant plasmid carrying genes for naphthalene degrading pathway

The stability of recombinant plasmid carrying genes for naphthalene mineralization was determined. A strain of Pseudomonas putida capable of mineralizing naphthalene (Nap⁺) via salicylate (Sal⁺) was isolated, and all regulatory and structural genes for the whole pathway were found to be encoded on a 25 kb Eco RI fragment of an approximately 83 kb plasmid present in this strain. The 25 kb Eco RI fragment was cloned into a tetracycline-resistant (Tc^R) cloning vector pLAFR3 and the recombinant plasmid, pRKJ3 (Nap⁺, Sal⁺, Tc^R), thus obtained was transferred into the plasmid-free strain Pseudomonas putida KT2442 in order to test the stability of the plasmid. Plasmid pRKJ3 was found to be segregationally and/or structurally unstable, depending on the growth conditions. Two types of novel derivative strains having the phenotypes Nap⁻, Sal⁺, Tc^R and Nap⁻, Sal⁻, Tc^R with specific deletions of approximately 2 kb and 18 kb, respectively, were obtained.     

5-N-Substituted-2-(substituted benzenesulphonyl) glutamines as antitumor agents. Part II: synthesis, biological activity and QSAR study

Cancer is a major killer disease throughout human history. Thus, cancer becomes a major point of interest in life science. It was proved that cancer is a nitrogen trap and tumor cells are avid glutamine consumers. The non-essential amino acid glutamine, which is a glutamic acid derivative, supplies its amide nitrogen to tumor cells in the biosynthesis of purine and pyrimidine bases of nucleic acids as well as takes part in protein synthesis. Based on these and in continuation of our composite programme of development of new potential anticancer agents through rational drug design, 17 new 5-N-Substituted-2-(substituted benzenesulphonyl) glutamines were selected for synthesis. These compounds as well as 36 earlier synthesized glutamine analogues were screened for antitumor activity using percentage inhibition of tumor cell count as the activity parameter. QSAR study was performed with 53 compounds in order to design leads with increased effectiveness for antitumor activity using both physicochemical and topological parameters. QSAR study showed that steric effect on the aromatic ring is conducive to the activity. n-butyl substitution on aliphatic side chain and atom no 12 is important for antitumor activity of glutamine analogues.     

C5L2 is a functional receptor for acylation-stimulating protein

C5L2 binds acylation-stimulating protein (ASP) with high affinity and is expressed in ASP-responsive cells. Functionality of C5L2 has not yet been demonstrated. Here we show that C5L2 is expressed in human subcutaneous and omental adipose tissue in both preadipocytes and adipocytes. In mice, C5L2 is expressed in all adipose tissues, at levels comparable with other tissues. Stable transfection of human C5L2 cDNA into HEK293 cells results in ASP stimulation of triglyceride synthesis (TGS) (193 +/- 33%, 5 microM ASP, p < 0.001, where basal = 100%) and glucose transport (168 +/- 21%, 10 microM ASP, p < 0.001). C3a similarly stimulates TGS (163 +/- 12%, p < 0.001), but C5a and C5a des-Arg have no effect. The ASP mechanism is to increase Vmax of glucose transport (149%) and triglyceride (TG) synthesis activity (165%) through increased diacylglycerolacyltransferase activity (200%). Antisense oligonucleotide down-regulation of C5L2 in human skin fibroblasts decreases cell surface C5L2 (down to 54 +/- 4% of control, p < 0.001, comparable with nonimmune background). ASP response is coordinately lost (basal TGS = 14.6 +/- 1.6, with ASP = 21.0 +/- 1.4 (144%), with ASP + oligonucleotides = 11.0 +/- 0.8 pmol of TG/mg of cell protein, p < 0.001). In mouse 3T3-L1 preadipocytes, antisense oligonucleotides decrease C5L2 expression to 69.5 +/- 0.5% of control, p < 0.001 (comparable with nonimmune) with a loss of ASP stimulation (basal TGS = 22.4 +/- 2.9, with ASP = 39.6 +/- 8.8 (177%), with ASP + oligonucleotides = 25.3 +/- 3.0 pmol of TG/mg of cell protein, p < 0.001). C5L2 down-regulation and decreased ASP response correlate (r = 0.761, p < 0.0001 for HSF and r = 0.451, p < 0.05 for 3T3-L1). In HEK-hC5L2 expressing fluorescently tagged beta-arrestin, ASP induced beta-arrestin translocation to the plasma membrane and formation of endocytic complexes concurrently with increased phosphorylation of C5L2. This is the first demonstration that C5L2 is a functional receptor, mediating ASP triglyceride stimulation.     

Biophysical properties and molecular characterization of amiloride-sensitive sodium channels in A549 cells

Amiloride-sensitive Na(+) channels, present in fetal and adult alveolar epithelial type II (ATII) cells, play a critical role in the reabsorption of fetal fluid shortly after birth and in limiting the extent of alveolar edema across the adult lung. Because of the difficulty in isolating and culturing ATII cells, there is considerable interest in characterizing the properties of ion channels and their response to injury of ATII cell-like cell lines such as A549 that derive from a human alveolar cell carcinoma. A549 cells were shown to contain alpha-, beta-, and gamma-epithelial Na(+) channel mRNAs. In the whole cell mode of the patch-clamp technique (bath, 145 mM Na(+); pipette, 145 mM K(+)), A549 cells exhibited inward Na(+) currents reversibly inhibited by amiloride, with an inhibition constant of 0.83 microM. Ion substitution studies showed that these channels were moderately selective for Na(+) (Na(+)-to-K(+) permeability ratio = 6:1). Inward Na(+) currents were activated by forskolin (10 microM) and inhibited by nitric oxide (300 nM) and cGMP. Recordings in cell-attached mode revealed the presence of an amiloride-sensitive Na(+) channel with a unitary conductance of 8.6 +/- 0.04 (SE) pS. Channel activity was increased by forskolin and decreased by nitric oxide and the cGMP analog 8-bromo-cGMP. These data demonstrate that A549 cells contain amiloride-sensitive Na(+) channels with biophysical properties similar to those of ATII cells.