Synthesis and biological activity of a new progestogen, 16-methylene-17alpha-hydroxy-18-methyl-19-norpregn-4-ene-3, 20-dione acetate

The progestational activity of second- and third-generation progestins in oral contraceptives were markedly increased by addition of an 18-methyl group. A new progestin, the 18-methyl analog of Nestorone, 16-methylene-17alpha-hydroxy-18-methyl-19-norpregn-4-ene-3,2 0-dione acetate (10), was synthesized. The relative binding affinity and biologic activity of 10 was compared with Nestorone, levonorgestrel, and progesterone using a binding assay for rat progesterone receptors, the Clauberg assay in the rabbit, and by assessing pregnancy maintenance in the rat. These studies, as summarized in Table 4, show that 10 is three to ten times more potent than Nestorone. The addition of the 18-methyl group to Nestorone markedly increased its potency as noted above, but is unlikely to change its rate of delivery from sustained release systems. 10 should be ideally suited for administration by implants or small skin patches.     

Structure-Activity Relationship and Mode of Action of a Frog Secreted Antibacterial Peptide B1CTcu5 Using Synthetically and Modularly Modified or Deleted (SMMD) Peptides

All life forms are equipped with rapidly acting, evolutionally conserved components of an innate immune defense system that consists of a group of unique and diverse molecules known as host defense peptides (HDPs). A Systematic and Modular Modification and Deletion (SMMD) approach was followed to analyse the structural requirement of B1CTcu5, a brevinin antibacterial peptide amide identified from the skin secretion of frog Clinotarsus curtipes, India, to show antibacterial activity and to explore the active core region. Seventeen SMMD-B1CTcu5 analogs were designed and synthesised by C and N-terminal amino acid substitution or deletion. Enhancement in cationicity by N-terminal Lys/Arg substitution or hydrophobicity by Trp substitution produced no drastic change in bactericidal nature against selected bacterial strains except S. aureus. But the sequential removal of N-terminal amino acids had a negative effect on bactericidal potency. Analog B1CTcu5-LIAG obtained by the removal of four N-terminal amino acids displayed bactericidal effect comparable to, or in excess of, the parent peptide with reduced hemolytic character. Its higher activity was well correlated with the improved inner membrane permeabilisation capacity. This region may act as the active core of B1CTcu5. Presence of C-terminal disulphide bond was not a necessary condition to display antibacterial activity but helped to promote hemolytic nature. Removal of the C-terminal rana box region drastically reduced antibacterial and hemolytic activity of the peptide, showing that this region is important for membrane targeting. The bactericidal potency of the D-peptide (DB1CTcu5) helped to rule out the stereospecific interaction with the bacterial membrane. Our data suggests that both the C and N-terminal regions are necessary for bactericidal activity, even though the active core region is located near the N-terminal of B1CTcu5. A judicious modification at the N-terminal region may produce a short SMMD analog with enhanced bactericidal activity and low toxicity against eukaryotic cells.     

Methotrexate Promotes Platelet Apoptosis via JNK-Mediated Mitochondrial Damage: Alleviation by N-Acetylcysteine and N-Acetylcysteine Amide

Thrombocytopenia in methotrexate (MTX)-treated cancer and rheumatoid arthritis (RA) patients connotes the interference of MTX with platelets. Hence, it seemed appealing to appraise the effect of MTX on platelets. Thereby, the mechanism of action of MTX on platelets was dissected. MTX (10 μM) induced activation of pro-apoptotic proteins Bid, Bax and Bad through JNK phosphorylation leading to ΔΨm dissipation, cytochrome c release and caspase activation, culminating in apoptosis. The use of specific inhibitor for JNK abrogates the MTX-induced activation of pro-apoptotic proteins and downstream events confirming JNK phosphorylation by MTX as a key event. We also demonstrate that platelet mitochondria as prime sources of ROS which plays a central role in MTX-induced apoptosis. Further, MTX induces oxidative stress by altering the levels of ROS and glutathione cycle. In parallel, the clinically approved thiol antioxidant N-acetylcysteine (NAC) and its derivative N-acetylcysteine amide (NACA) proficiently alleviate MTX-induced platelet apoptosis and oxidative damage. These findings underpin the dearth of research on interference of therapeutic drugs with platelets, despite their importance in human health and disease. Therefore, the use of antioxidants as supplementary therapy seems to be a safe bet in pathologies associated with altered platelet functions.     

Is AKR2A an essential molecular chaperone for a class of membrane-bound proteins in plants?

The Arabidopsis ankyrin-repeat containing protein 2A (AKR2A) was shown to be an essential molecular chaperone for the peroxisomal membrane-bound ascorbate peroxidase 3 (APX3), because the biogenesis of APX3 depends on the function of AKR2A in plant cells. AKR2A binds specifically to a sequence in APX3 that is made up of a transmembrane domain followed by a few positively charged amino acid residues; this sequence is named as AKR2A-binding sequence or ABS. Interestingly, a sequence in the chloroplast outer envelope protein 7 (OEP7) shares similar features to ABS and is able to bind specifically to AKR2A, suggesting a possibility that proteins with a sequence similar to ABS could bind to AKR2A and they are all likely ligand proteins of AKR2A. This hypothesis was supported by analyzing five additional proteins that contain sequences similar to ABS using the yeast two-hybrid technique. A preliminary survey in the Arabidopsis genome indicates that there are at least 500 genes encoding proteins that contain sequences similar to ABS, which raises interesting questions: are these proteins AKR2A''s ligand proteins and does AKR2A play a critical role in the biogenesis of these proteins in plants?Key words: Arabidopsis, membrane protein, molecular chaperone, protein targeting, transmembrane domainThe Arabidopsis ankyrin-repeat containing protein 2A (AKR2A) is an essential molecular chaperone for the peroxisomal membrane-bound ascorbate peroxidase 3 (APX3).¹ Both AKR2A and APX3 were identified as GF14λ-interacting proteins^2,3 when the mode of action of a 14-3-3 protein, GF14λ⁴ was studied. In characterizing the enzymatic property of APX3, there was some initial difficulty in purifying the expressed APX3 from a bacterial expression system. Although APX3 could be expressed in E. coli cells in large quantities, as evidenced by directly boiling the bacterial cells and analyzing the bacterial cells by SDS-PAGE and Western blot analysis (Fig. 1), APX3 enzymatic activity in the supernatant fraction was not detectable after cells were broken by sonication (Fig. 1). The reason that APX3 activity was not detectable in the supernatant fraction was likely caused by the transmembrane domain that occurs at the C-terminal end of APX3; because these hydrophobic domains could interact with one another, forming insoluble aggregates in bacterial cells. When a truncated APX3 was expressed, i.e., APX3 without the transmembrane domain (APX3Δ in Fig. 1), APX3 activity was then detectable in the supernatant fraction of bacterial cellular extracts. If a protein is able to bind to APX3''s transmembrane domain immediately after or during translation of APX3, this protein could prevent APX3 from forming insoluble aggregates among themselves. APX3 activity would then be detectable in the supernatant fraction. Because some 14-3-3-interacting proteins were shown to interact with one another,⁵ the best candidate that could interact with APX3 should be AKR2A (because they are both GF14λ-interacting proteins). This possibility was tested by simultaneously expressing both APX3 and AKR2A in the same bacterial cell; APX3 activity was indeed detectable in the supernatant fraction of bacterial cellular extracts (Fig. 1).Open in a separate windowFigure 1Protein-protein interaction between AKR2A and APX3 in bacterial cells. (A) Analysis of APX3 activity in supernatant fractions of various bacterial cells. In lanes, APX3, supernatant from cells that express full-length APX3; APX3 + OMT 1, supernatant from cells that express both full-length APX3 and OMT 1 (O-methyltransferase1,⁷); APX3 + AKR2A, supernatant from cells that express both full-length APX3 and AKR2A; APX3Δ, supernatant from cells that express a partial APX3 (i.e., lacking the transmembrane domain and the last seven amino acid residues); APX3Δ + OMT 1, supernatant from cells that express both APX3Δ and OMT 1; APX3Δ + AKR2A, supernatant from cells that express both APX3Δ and AKR2A; OMT 1, supernatant from cells that express OMT1; AKR2A, supernatant from cells that express AKR2A. The white bands in the gel represent APX3 activities as assayed by using the method of Mittler and Zilinskas.⁸ (B) Bacterial cells expressing various target proteins were analyzed directly by using SDS-PAGE method and the positions of the expressed target proteins are marked on the right. (C) Bacterial cells expressing various target proteins were analyzed by western blot. The antibodies used are listed on the right.This was the first evidence that AKR2A interacts with APX3 and the interaction site involves the C-terminal transmembrane domain of APX3. To further define the amino acid residues involved in the AKR2A-APX3 interaction, yeast two-hybrid experiments were conducted with various deletion fragments of AKR2A and APX3.1 It was found that in addition to the transmembrane domain, the positively charged amino acid residues following the transmembrane domain also play a role in the AKR2A-APX3 interaction.¹ This sequence in APX3 was designated as AKR2A-binding sequence (ABS). In order to understand the biological function of the AKR2A-APX3 interaction, several akr2a mutants that displayed reduced or altered interaction with APX3 were created and analyzed. Results indicated that reduced AKR2A activity leads to severe developmental, phenotypic, and physiological abnormalities including reduced steady-state level of APX3 and reduced targeting of APX3 to peroxisomal membranes in Arabidopsis.¹ The pleiotropic nature of akr2a mutants indicated that AKR2A plays more roles in addition to chaperoning APX3. Indeed this work was corroborated by a finding that AKR2A is also required for the biogenesis of the chloroplast outer envelope protein 7 (OEP7).⁶ More importantly, the interaction between AKR2A and OEP7 also involves a sequence in OEP7 that is similar to the ABS found in APX3.There is no apparent similarity, at the amino acid level, between the sequences of the AKR2A-binding site found in APX3 and OEP7; it appears that what AKR2A recognizes in its ligand proteins is the structural feature: single transmembrane domain followed by one or a few positively charged amino acid residues. Therefore, these AKR2A-binding sequences should all be designated as ABS, and it was predicted that any protein with an ABS could be AKR2A''s interacting protein. Five such proteins, APX5, TOC34, TOM20, cytochrome b5 (CB5) and cytochrome b₅ reductase (CB5R) were tested, and indeed all five proteins interacted with AKR2A in the yeast two-hybrid system.¹ More importantly, the interaction sites of these proteins are their ABS in every case tested.¹ Based on these discoveries, it is proposed that AKR2A is a molecular chaperone for this group of ABS-containing proteins.Among the seven AKR2A-interacting proteins that were characterized, the ABS is found at C-terminal end of four proteins (APX3, APX5, CB5 and TOM20), near N-terminal end of two proteins (OEP7 and CB5R), and near C-terminal end of one protein (TOC34), suggesting that the position of ABS in these membrane proteins does not affect its interaction with AKR2A. Furthermore, in all cases, AKR2A binds to its ligand proteins that contain only one ABS. AKR2A does not appear to bind to proteins that contain multiple transmembrane domains such as PMP22,^1,6 even though these transmembrane domains are followed by a few positively charged amino acid residues.APX3 and APX5 are peroxisomal membrane-bound, OEP7 and TOC34 are chloroplast outer envelope proteins, TOM20 is a mitochondrion outer membrane protein and CB5 and CB5R are microsomal membrane (ER-membrane) proteins. Therefore, AKR2A is clearly not responsible for targeting these proteins to their specific membranes; instead AKR2A serves as a molecular chaperone to prevent these proteins from forming aggregates through their hydrophobic domain in ABS after translation (Fig. 2). Perhaps, AKR2A''s binding to the ABS of these membrane proteins also keeps these proteins in insertion competent state before they are sent to their specific destinations. It is clear that other factors, such as organellar membrane-specific receptors, must be required for sending these proteins to their specific membranes (Fig. 2).Open in a separate windowFigure 2Model on how AKR2A chaperones its ligand proteins. (1) AKR2A binds to ABS of a nascent protein that is being synthesized from a free ribosome. (2) AKR2A keeps its ligand protein (L) in the cytoplasm. (3) With the help of membrane-specific receptors, AKR2A''s ligand proteins are sent to their specific membranes.The Arabidopsis proteome was analyzed and it was found that there are at least 500 proteins that contain sequences similar to ABS (http://bio.scu.edu.cn/list.xls). Would these proteins be AKR2A''s ligand proteins? Some of them, if not all, might be, but it will be a challenging task to experimentally test these proteins one by one. A better bioinformatics tool that can provide clues on the mode of action of the protein-protein interactions between AKR2A and its known ligand proteins should help us designing next set of experiments in order to answer the above question in an efficient way.     

Baculovirus expression system: An alternative for producing catalytically active human PTP-1B

Protein tyrosine phosphatases (PTPs) play multiple roles in many physiological processes. Over-expression of the PTPs has been shown to be associated with cellular toxicity, which may also lead to the deletion of the respective gene from stable cell clones. We also observed that PTP-1B over-expression in CHO and HEK293 stable cell clones led to cytotoxicity and low revival rates during clone generation and maintenance. To address these issues, bacmid transposition technology was utilized to generate recombinant PTP-1B baculovirus, and Spodoptera frugiperda (Sf9 and Sf21) insect cell lines were infected with the virus. The data obtained on expression and activity of the PTP-1B highlights clear advantage of the recombinant baculovi-rus-insect cell expression system over the mammalian cell line technique due to increase in enzyme activity, strongly inhibited by phosphatase specific inhibitor RK682. Possible application of the expression system for producing active enzymes in bulk quantity for a new drug discovery is also discussed.     

Biodegradation and corrosion behaviour of <Emphasis Type="Italic">Serratia marcescens</Emphasis> ACE2 isolated from an Indian diesel-transporting pipeline

A facultative anaerobic species Serratia marcescens ACE2 isolated from the corrosion products of a diesel-transporting pipeline in North West India was identified by 16S rDNA sequence analysis. The role of Serratia marcesens ACE2 on biodegradation of commercial corrosion inhibitor (CCI) and its influence on the corrosion of API 5LX steel has been enlightened. The degrading strain ACE2 is involved in the process of corrosion of steel API 5LX and also utilizes the inhibitor as organic source. The quantitative biodegradation efficiency of corrosion inhibitor was 58%, which was calculated by gas chromatography mass spectrum analysis. The effect of CCI on the growth of bacteria and its corrosion inhibition efficiency were investigated. Additionally, the role of this bacterium in corrosion of steel has been investigated by powder X-ray diffractometer (XRD) and scanning electron microscope studies. The presence of high-intensity ferric oxides and manganese oxides noticed from the XRD indicates that ACE2 enhances the corrosion process in presence of inhibitor as a carbon source. This basic study will be useful for the development of new approaches for the detection, monitoring and control of microbial corrosion in petroleum product pipelines.     

Tyrosine sulfation of statherin

Tyrosylprotein sulfotransferase (TPST), responsible for the sulfation of a variety of secretory and membrane proteins, has been identified and characterized in submandibular salivary glands (William et al. Arch Biochem Biophys 1997; 338: 90-96). In the present study we demonstrate the sulfation of a salivary secretory protein, statherin, by the tyrosylprotein sulfotransferase present in human saliva. Optimum statherin sulfation was observed at pH 6.5 and at 20 mm MnCl(2). Increase in the level of total sulfation was observed with increasing statherin concentration. The K(m)value of tyrosylprotein sulfotransferase for statherin was 40 microM. Analysis of the sulfated statherin product on SDS-polyacrylamide gel electrophoresis followed by autoradiography revealed (35)S-labelling of a 5 kDa statherin. Further analysis of the sulfated statherin revealed the sulfation on tyrosyl residue. This study is the first report demonstrating tyrosine sulfation of a salivary secretory protein. The implications of this sulfation of statherin in hydroxyapatite binding and Actinomyces viscosus interactions are discussed.