Use of micelles in studying drug-binding sites: simulation of the tubulin-bound fluorescence of colchicine

Enhancement of the fluorescence intensity of colchicine occurs in media of low polarity and appreciable viscosity; this is suggested to be the basis of the intensification of its fluorescence when it is bound to and immobilized in tubulin. We show here that the tubulin-bound fluorescence features of colchicine are largely reconstructed upon solubilizing it in chosen micellar aggregates that offer optimal polarities and microviscosities. Triton X-100 and bile salt micelles intensify the colchicine emission but the maximal effects are obtained with tetrameric aggregates of the peptide melittin. Estimates of the polarity, microviscosity and binding-site dimensions of colchicine are obtained using this mimetic approach. Our results suggest that well chosen micellar systems act as good models to reconstruct and analyze the spectral properties of molecules immobilized in their binding sites.     

RAB-6.1 and RAB-6.2 Promote Retrograde Transport in C. elegans

Retrograde transport is a critical mechanism for recycling certain membrane cargo. Following endocytosis from the plasma membrane, retrograde cargo is moved from early endosomes to Golgi followed by transport (recycling) back to the plasma membrane. The complete molecular and cellular mechanisms of retrograde transport remain unclear. The small GTPase RAB-6.2 mediates the retrograde recycling of the AMPA-type glutamate receptor (AMPAR) subunit GLR-1 in C. elegans neurons. Here we show that RAB-6.2 and a close paralog, RAB-6.1, together regulate retrograde transport in both neurons and non-neuronal tissue. Mutants for rab-6.1 or rab-6.2 fail to recycle GLR-1 receptors, resulting in GLR-1 turnover and behavioral defects indicative of diminished GLR-1 function. Loss of both rab-6.1 and rab-6.2 results in an additive effect on GLR-1 retrograde recycling, indicating that these two C. elegans Rab6 isoforms have overlapping functions. MIG-14 (Wntless) protein, which undergoes retrograde recycling, undergoes a similar degradation in intestinal epithelia in both rab-6.1 and rab-6.2 mutants, suggesting a broader role for these proteins in retrograde transport. Surprisingly, MIG-14 is localized to separate, spatially segregated endosomal compartments in rab-6.1 mutants compared to rab-6.2 mutants. Our results indicate that RAB-6.1 and RAB-6.2 have partially redundant functions in overall retrograde transport, but also have their own unique cellular- and subcellular functions.     

Development of sporeless/low sporing strains of Pleurotus through mutation

Summary The sporophores of Pleurotus are gymnocarpous and continuously release spores in the atmosphere causing respiratory allergies like hay fever and farmer’s lung disease among workers. The allergy is caused by the antigens present on the walls of the spores. Apart from this, during commercial production, these spores settle on the fruit bodies, germinate and form a velvety film which gives an unpleasant appearance to the mushrooms. The spores emitted may include new genotypes likely to attack wood or trees. Spore allergy is one of the most important limiting factors for the large scale cultivation of this species. Different approaches are being adopted at IIHR for the production of commercial sporeless/low-sporing strains of Pleurotus to alleviate the spore allergy problem. Attempts were made during the present investigation to produce sporeless or low-sporing mutants through u.v. mutation. Mutation of the mycelium did not yield the desired results. Mutation of the spores of Pleurotus sajor-caju yielded an extremely low-sporing mutant after 75 min exposure. The character has been found to be stable for more than 10 generations of subculturing.     

Origin of life: A hypothesis for the origin of adaptor-mediated ordered synthesis of proteins and an explanation for the choice of terminating codons in the genetic code

Life can be defined as a system of self-sustained chemical processes springing from the ordered synthesis of proteins directed by nucleic acids. To the notoriously difficult problem of the origin of this basic process of nucleic acid-directed protein synthesis, we give a solution of molecular interactions between pentanucleotides and amino acids. A particular conformation of a pentanucleotide forms a double sided template, with its ‘inside’ capable of nestling an amino acid while the ‘outside’ acts as an adaptor to a ‘codon’ triplet on long-chain nucleic acids. This serves as a primitive decoding system. An important aspect of our postulate is that a dynamic interaction is triggered, by this decoding system, through which amino acids are brought to juxtaposition facilitating peptide bond formation. Almost all the important and unique features of contemporary protein-synthesizing machinery are seen to be a direct and natural consequence of our postulate. The emergence of the termination codons also fits in, as a natural consequence of this molecular mechanism.     

Mammalian sulfoconjugate metabolism

Sulfoconjugates occur ubiquitously as sulfopolysaccharides, sulfolipids and sulfoproteins. A variety of sulfotransferases catalyze the sulfation process with 3’-phosphoadenosine 5’-phosphosulfate as the sulfate donor. Sulfatases that catalyze the desulfation of different sulfoconjugates are known to be deficient in a number of genetic storage disorders.     

Effects of prolactin and androgens on seminal vesicular lipids of castrated mature bonnet monkeys Macaca radiata

Effects of prolactin (PRL), bromocriptine (Br), testosterone propionate (TP), dihydrotestosterone (DHT) and the combination of these androgens with PRL/Br on the total lipid, total cholesterol, total glyceride glycerols, total phospholipid and their fractions in seminal vesicles of castrated mature monkeys were studied. Glyceride glycerols formed the major portion (50%) of total lipids in normal monkeys. Cholesterol and phospholipids were of equal share (25%). Esterified cholesterol formed major share (75%) of total cholesterol. Diacyl glycerol was the major (60%) glyceride glycerol and phosphatidyl choline and ethanolamine were the major phospholipid classes. Except triacyl glycerol castration markedly decreased all the lipid classes. PRL restored normal free and esterified cholesterol and phosphatidyl inositol but Br invariably decreased all the lipid classes. TP/DHT treatment stimulated the free and esterified cholesterol more than the control; it restored the normal glyceride glycerols. Phosphatidyl inositol, choline and ethanolamine were stimulated by androgens and other phospholipid classes were brought to normal. Addition of PRL + TP/DHT markedly increased esterified cholesterol, phosphatidyl inositol, choline, ethanolamine and phosphatidic acid. In all these aspects, Br counteracted the effects of androgens and PRL.     

TGF-Beta inhibits the in vitro induction of lymphokine-activated killing activity

Summary Employing serum-free media, human peripheral blood mononuclear cells, and purified recombinant interleukin-2 (IL-2), conditions were observed in which the development of IL-2-driven cytotoxic activity was suppressed. The cytotoxic activity of such IL-2-generated lymphokine activated killing (LAK) was tested against natural killer-resistant cultured tumor cells (Daudi, Raji, and a glioma). LAK generation was inhibited by addition of some normal sera, normal platelets, or some tumor cells. Because recent reports have indicated that transforming growth factor-beta (TGF-beta)-like factors are often secreted by tumors and the acidic alpha granules of platelets and can be present in sera, we tested the effect of purified human TGF-beta on the activation of LAK. Our results indicated that TGF-beta is very suppressive for LAK induction, and can completely prevent both the IL-2-driven proliferation and cytotoxicity at concentrations as low as 5 ng/ml. Titrations of IL-2 and of TGF-beta indicated that the suppression is dose-dependent and can be avoided by employing higher levels of IL-2. It was also found that the suppressive effect of TGF-beta can be overcome by washing suppressed cell populations and further culture in low levels of IL-2. Collectively, these data indicate that TGF-beta can be a potent inhibitor of LAK generation under standard activation conditions, but that this effect is regulated by the relative level of IL-2 and may be overcome and/or reversed in vitro.     

Chemical modification of the bifunctional human serum pseudocholinesterase. Effect on the pseudocholinesterase and aryl acylamidase activities

The effect of chemical modification on the pseudocholinesterase and aryl acylamidase activities of purified human serum pseudocholinesterase was examined in the absence and presence of butyrylcholine iodide, the substrate of pseudocholinesterase. Modification by 2-hydroxy-5-nitrobenzyl bromide, N-bromosuccinimide, diethylpyrocarbonate and trinitrobenzenesulfonic acid caused a parallel inactivation of both pseudocholinesterase and aryl acylamidase activities that could be prevented by butyrylcholine iodide. With phenylglyoxal and 2,4-pentanedione as modifiers there was a selective activation of pseudocholinesterase alone with no effect on aryl acylamidase. This activation could be prevented by butyrylcholine iodide. N-Ethylmaleimide and p-hydroxy-mercuribenzoate when used for modification did not have any effect on the enzyme activities. The results suggested essential tryptophan, lysine and histidine residues at a common catalytic site for pseudocholinesterase and aryl acylamidase and an arginine residue (or residues) exclusively for pseudocholinesterase. The use of N-acetylimidazole, tetranitromethane and acetic anhydride as modifiers indicated a biphasic change in both pseudocholinesterase and aryl acylamidase activities. At low concentrations of the modifiers a stimulation in activities and at high concentrations an inactivation was observed. Butyrylcholine iodide or propionylcholine chloride selectively protected the inactivation phase without affecting the activation phase. Protection by the substrates at the inactivation phase resulted in not only a reversal of the enzyme inactivation but also an activation. Spectral studies and hydroxylamine treatment showed that tyrosine residues were modified during the activation phase. The results suggested that the modified tyrosine residues responsible for the activation were not involved in the active site of pseudocholinesterase or aryl acylamidase and that they were more amenable for modification in comparison to the residues responsible for inactivation. Two reversible inhibitors of pseudocholinesterase, namely ethopropazine and imipramine, were used as protectors during modification. Unlike the substrate butyrylcholine iodide, these inhibitors could not protect against the inactivation resulting from modification by 2-hydroxy-5-nitrobenzyl bromide, N-bromosuccinimide and trinitrobenzenesulfonic acid. But they could protect against the activation of pseudocholinesterase and aryl acylamidase by low concentrations of N-acetylimidazole and acetic anhydride thereby suggesting that the binding site of these inhibitors involves the non-active-site tyrosine residues.