Natural products and their derivatives as inhibitors of glycogen phosphorylase: potential treatment for type 2 diabetes

Glycogen phosphorylase (GP) (EC 2.4.1.1) is an important therapeutic target for the potential treatment of type 2 diabetes. The search for potent, selective and drug-like GP inhibitors which may eventually lead to hypoglycaemic agents has to date uncovered a number of natural product inhibitors with both pharmaceutical and nutraceutical potential. GP is an allosteric protein with at least six different ligand binding sites that modulate its enzymatic activity. Hence, inhibitors with considerable structural diversity can be designed. This review is focused on advances in the discovery of natural products and their derivatives as GP inhibitors.     

Synthetic constructs functionally mimicking ribonuclease A

The mechanism of hydrolysis of RNA substrates—diribonucleoside monophosphate CpA and decaribonucleotide UUCAUGUAAA—by chemical constructs functionally mimicking ribonuclease A was studied. It is shown that RNA cleavage by chemical RNases 2L2 and 2D3 proceeds similar to the RNase A-induced RNA hydrolysis through 2′,3′-cyclophosphate as an intermediate product. A comparison of hydrolyses of CpA in water and D₂O revealed an isotope effect (K _H/K _D=2.28), which implies acid-base catalysis at the limiting stage of the reaction. Two feasible mechanisms of RNA hydrolysis by chemical RNases (linear and adjacent) are discussed.     

The Role of Low-Molecular-Weight Nitrogen Compounds in the Osmotolerance of Rhodococcus erythropolis and Arthrobacter globiformis

Investigations showed that Rhodococcus erythropolis E-15 and Arthrobacter globiformis 2F cells respond to osmotic shock by increasing the synthesis of free amino acids, primarily glutamic acid (80% of the intracellular free amino acid pool). The osmoprotective role of glutamic acid follows from its beneficial effect on the growth of bacteria in high-salinity media. It was found that the addition of this amino acid to the growth medium at a concentration of 2 mM shortened the lag phase and increased the growth rate and biomass yield of either of the two bacteria. The addition of another osmoprotectant, trehalose, to the high-salinity growth medium of R. erythropolis E-15 at the same concentration (2 mM), restored the growth parameters of this bacterium to the control values.     

Affinity of guanosine derivatives for polycytidylate revisited

Evidence is presented for complexation of guanosine 5-monophosphate 2-methylimidazolide (2-MeImpG) with polycytidylate (poly(C)) at pH 8.0 and 23°C in the presence of 1.0 M NaCl and 0.2 M MgCl₂ in water. The association of 2-McImpG with poly(C) was investigated using UV-vis spectroscopy as well as by monitoring the kinetics of the nucleophilic substitution reaction of the imidazole moiety by amines. The results of both methods are consistent with moderately strong poly(C) · 2-McImpG complexation and the spectrophotometric measurements allowed the construction of a binding isotherm with a concentration of 2-McImpG equal to 5.55 ± 0.15 mM at half occupancy. UV spectroscopy was employed to establish the binding of other guanosine derivatives on poly(C). These derivatives are guanosine 5-monophosphate (5GMP), guanosine 5monophosphate imidazolide (ImpG), and guanosine 5monophosphate morpholidate (morpG). Within experimental error these guanosine derivatives exhibit the same affinity for poly(C) as 2-McImpG.     

DIMERIZATION IN HIGHLY CONCENTRATED SOLUTIONS OF PHOSPHOIMIDAZOLIDE ACTIVATED MONONUCLEOTIDES

Phosphoimidazolide activated ribomononucleotides (*pN) are useful substrates for the non-enzymatic synthesis of polynucleotides. However, dilute neutral aqueous solutions of *pN typically yield small amounts of dimers and traces of polymers; most of *pN hydrolyzes to yield nucleoside 5-monophosphate. Here we report the self-condensation of nucleoside 5-phosphate 2-methylimidazolide (2-MeImpN with N = cytidine, uridine or guanosine) in the presence of Mg2+ in concentrated solutions, such as might have been found in an evaporating lagoon on prebiotic Earth. The product distribution indicates that oligomerization is favored at the expense of hydrolysis. At 1.0 M, 2-MeImpU and 2-MeImpC produce about 65% of oligomers including 4% of the 3,5-linked dimer. Examination of the product distribution of the three isomeric dimers in a self-condensation allows identification of reaction pathways that lead to dimer formation. Condensations in a concentrated mixture of all three nucleotides (U,C,G mixtures) is made possible by the enhanced solubility of 2-MeImpG in such mixtures. Although percent yield of internucleotide linked dimers is enhanced as a function of initial monomer concentration, pyrophosphate dimer yields remain practically unchanged at about 20% for 2-MeImpU, 16% for 2-MeImpC and 25% of the total pyrophosphate in the U,C,G mixtures. The efficiency by which oligomers are produced in these concentrated solutions makes the evaporating lagoon scenario a potentially interesting medium for the prebiotic synthesis of dimers and short RNAs.     

A review of sciarids of the genus <Emphasis Type="Italic">Leptosciarella</Emphasis> Tuomikoski 1960 (Diptera,Sciaridae) in the Altai fauna,with description of three new species

The taxonomy of the genus Leptosciarella Tuomikoski, 1960 in the Altai Area within Altai Territory and the Altai Republic was studied. Eleven species were recognized, including three new species which are described and illustrated here: Leptosciarella (L.) ulagana Komarova sp. n., L. (L.) angustistylus Komarova sp. n., L. (L.) senex Komarova sp. n. Additionally, L. (L.) fuscipalpa (Mohrig et Mamaev 1979) and L. (L.) truncata (Tuomikoski 1960) are recorded from Altai for the first time. A key to the Altai species is provided.     

The protocol for the isolation and cryopreservation of osteoclast precursors from mouse bone marrow and spleen

Osteoclasts are responsible for physiological bone remodeling as well as pathological bone destruction in osteoporosis, periodontitis and rheumatoid arthritis, and thus represent a pharmacological target for drug development. We aimed to characterize and compare the cytokine-induced osteoclastogenesis of bone marrow and spleen precursors. Established protocols used to generate osteoclasts from bone marrow were modified to examine osteoclastogenesis of the spleen cells of healthy mice. Osteoclast formation was successfully induced from spleen precursors using receptor activator of nuclear factor κB ligand (50 ng/ml) and macrophage colony stimulating factor (50 ng/ml). Compared to bone marrow cultures, differentiation from spleen required a longer cultivation time (9 days for spleen, as compared to 5 days for marrow cultures) and a higher plating density of non-adherent cells (75,000/cm² for spleen, as compared to 50,000/cm² for bone marrow). Osteoclasts generated from spleen precursors expressed osteoclast marker genes calcitonin receptor, cathepsin K and matrix metalloproteinase 9 and were capable of resorbing hydroxyapatite. The differentiation capacity of spleen and bone marrow precursors was comparable for BALB/c, C57BL/6 and FVB mice. We also developed and tested a cryopreservation protocol for the osteoclast precursors. While 70–80 % of cells were lost during the first week of freezing, during the subsequent 5 weeks the losses were within 2–5 % per week. Osteoclastogenesis from the recovered bone marrow precursors was successful up to 5 weeks after freezing. Spleen precursors retained their osteoclastogenic capacity for 1 week after freezing, but not thereafter. The described protocol is useful for the studies of genetically modified animals as well as for screening new osteoclast-targeting therapeutics.     

Synthesis and analysis of conformationally restricted analogs of peptide inhibitors of the angiotensin-converting enzyme

To investigate conformations of peptide inhibitors of the angiotensin-converting enzyme in the enzyme-inhibitor complex, the synthesis, studies of inhibitory activity, and conformational calculations of analogues of bradykinin-potentiating peptides with N-methylalanine or D-alanine in place of L-proline or L-alanine residues have been carried out. All the analogues showed a sharp decrease of inhibitory activity in comparison with the natural peptides, that might be considered as an indirect confirmation of the earlier proposed "conformation of inhibition" of the above-mentioned peptides.     

Role of emulsification in the process of hydrocarbon absorption by Pseudomonas aeruginosa cells

Lipophilic biopolymers from the cell walls of saprophytic mycobacteria were shown to stimulate the process of hydrocarbon assimilation by Pseudomonas aeruginosa cells. This should be attributed to the fact that bacterial peptidoglycolipids emulsify a hydrocarbon facilitating the contact between it and the cells. It has been found experimentally that P. aeruginosa cells growing in the medium with n-alkanes release a factor into the medium. The factor appears to contain peptide chains and is responsible for hydrocarbon emulsification.