α-Amylase structure and activity

Two computerized methods of predicting protein secondary structure from amino acid sequences are evaluated by using them on the α-amylase ofAspergillus oryzae, for which the three-dimensional structure has been determined. The methods are then used, with amino acid alignments, to predict the structures of other α-amylases. It is found that all α-amylases of known amino acid sequence have the same basic structure, a barrel of eight parallel stretches of extended chain surrounded by eight helices. Strong similarities are found in those areas of the proteins believed to bind an essential calcium ion and at that part of the active site that catalyzes bond hydrolysis in the substrates. The active site, as a whole, is formed mainly of amino acids situated on loops joining extended chain to the adjacent helix. Variations in the length and amino acid sequence of these loops, from one α-amylase to another, provide the differences in binding the substrates believed to account for the known variations in action pattern of α-amylases of different biological origins.     

Distribution and biological activity ofβ-thymosins

Summary -Thymosins, a group of highly homologous peptides consisting of about 40 amino acid residues, were found to be distributed from mammals up to echinoderms. Althogh they have first been isolated from mammalian thymus tissue preparations, their occurrance is not organ-specific and they are present even in different types of cells. For thymosin ₄ several biological activities have been reported, stating that this peptide acts as a thymus peptide hormone and is also involved in the neuroendocrine and immune system. However, it was recently demonstrated that thymosin ₄ has actin-sequestering properties and therefore might play an important role in the regulation of the microfilament system. This fact gives a new outlook on the real biological function of-thymosins.     

Chromosome aberration and environmental physical activity: Down syndrome and solar and cosmic ray activity, Israel, 1990–2000

The possibility that environmental effects are associated with chromosome aberrations and various congenital pathologies has been discussed previously. Recent advances in the collection and computerization of data make studying these potential associations more feasible. The aim of this study was to investigate a possible link between the number of Down syndrome (DS) cases detected prenatally or at birth yearly in Israel over a 10-year period compared with the levels of solar and cosmic ray activity 1 year before the detection or birth of each affected child. Information about 1,108,449 births was collected for the years 1990–2000, excluding 1991, when data were unavailable. A total of 1,310 cases of DS were detected prenatally or at birth—138 in the non-Jewish community and 1,172 in the Jewish population. Solar activity indices—sunspot number and solar radio flux 2,800 MHz at 10.7 cm wavelength for 1989–1999—were compared with the number of DS cases detected. Pearson correlation coefficients (r) and their probabilities (P) were established for the percentage of DS cases in the whole population. There was a significant inverse correlation between the indices of solar activity and the number of cases of DS detected—r=–0.78, P=0.008 for sunspot number and r=–0.76, P=0.01 for solar flux. The possibility that cosmophysical factors inversely related to solar activity play a role in the pathogenesis of chromosome aberrations should be considered. We have confirmed a strong trend towards an association between the cosmic ray activity level and the incidence of DS.     

Synthesis,characterization, antimicrobial activity and structure–activity relationships of new aryldisulfonamides

A series of aromatic disulfonamide (1-8) derivatives and 4-methylbenzenesulfonyl hydrazide (9) were synthesized and characterized. All compounds were evaluated in vitro for their antimicrobial activity against Staphylococcus aureus ATCC 25953, Bacillus cereus ATCC 6633, Bacillus magaterium RSKK 5117, Escherichia coli ATCC 11230, Salmonella enterititis ATCC 13076 by microdilution and disc diffusion methods. Antimicrobial activity of the aromatic disulfonamides decreased as the length of the carbon chain increased. An analysis of the structure- activity relationship (SAR) along with computational studies showed that the most active compound (9) possessed low lipophilicity (AlogP=0.59) and high solubility (logS = -1.33).     

Synthesis and anticancer activity of quinopimaric and maleopimaric acids’ derivatives

A series of quinopimaric and maleopimaric acids’ derivatives modified in the E-ring, at the carbonyl- and carboxyl-groups were synthesized and their in vitro cytotoxic activity was evaluated at the National Cancer Institute, USA. Methyl esters of dihydroquinopimaric, 1a,4a-dehydroquinopimaric, 2,3-epoxyquinopimaric, 1-ethylenketal-dihydroquinopimaric, 1-ethylenketal-4-hydroxyiminodihydroquinopimaric acids displayed an activity on renal cancer, leukemia, colon cancer and breast cancer cell lines in concentration 10⁻⁵ M. Methyl 1,4-dihydroxyiminodihydroquinopimarate showed both a potent and broad spectrum of cytotoxic activity against NSC lung cancer, colon cancer, breast cancer, renal cancer and leukemia and revealed in vivo antineoplastic activity towards mouse solid transplantable mammary carcinoma Ca755 and colon adenocarcinoma AKATOL. The information about antineoplastic activity of the studied quinopimaric and maleopimaric acids’ derivatives will be used for hit to lead optimization in these chemical series.     

Hyperlipidemia and type I 5′-monodeiodinase activity

Male New Zealand White rabbits were divided into three groups: (I) control, (II) high-fat-diet (HFD) fed, and (III) HFD fed+selenium supplemented. After 3 mo of treatment, there was a significant increase in serum cholesterol and triglycerides in the HFD-fed group as compared to the control. However in the selenium (Se)-supplemented group, the levels of serum cholesterol and triglycerides were significantly less as compared to group II. HFD feeding resulted in decreased serum Se levels, but supplementation of dietary Se along with HFD, as in group III, showed an apparent increase in its levels. The Se-dependent glutathione peroxidase (GSH-Px) activity in the liver and the aorta increased significantly in HFD-fed animals and also showed an additional significant increase on Se supplementation. Both serum T₃ and T₄ levels showed a significant decrease on HFD feeding. However, supplementation of Se led to a significant increase in the levels of these parameters viz-à-viz HFD-fed animals. HFD feeding significantly decreased the activity of type I iodothyronine 5′-deiodinase (5′-DI) in the liver from group II rats. On supplementation of Se along with HFD, the activity increased in the liver. However, there was no significant change in its activity in the aorta. The 5′-DI activity in the thyroid showed an opposite trend in comparison with peripheral tissues (i.e., liver). The important finding of this study is that in the hyperlipidemic state, deiodinase in the thyroid behaves in a different manner as compared to its activity in extrathyroidal tissues.     

Antibacterial activity of Δ9-tetrahydrocannabinol and cannabidiol

The minimum inhibiting concentrations (MIC) of ⁹-tetrahydrocannabinol (THC) and cannabidiol (CBD) for staphylococci and streptococci in broth are in the range of 1–5 g/ml. In the same range, both compounds are also bactericidal. In media containing 4% serum or 5% blood the antibacterial activity is strongly reduced (MIC 50g/ml). Gram-negative bacteria are resistant to THC and CBD.     

Synthesis,antibiotic activity and structure–activity relationship study of some 3-enaminetetramic acids

The synthesis and evaluation of 3-enaminetetramic acids as antibacterial agents is reported; contrary to the analogous 3-acyltetramic acids, the enaminetetramic acid class of compound exhibits modest antibacterial activity against a limited spectrum of organisms, and even that activity is strongly dependent on the identity of the tetramate ring substituents. Moreover, these compounds appear to have a different mode of action to the analogous 3-acyltetramic acids, and appear to offer more limited opportunity for further elaboration in drug discovery.     

Synthesis and antiproliferative activity of novel α- and β-dialkoxyphosphoryl isothiocyanates

A series of 15 mostly new dialkoxyphosphoryl alkyl and aralkyl isothiocyanates were synthesized using two alternative strategies, and their in vitro antiproliferative activity against several cancer cell lines (including drug resistant) is here demonstrated. The IC₅₀ values measured for the new compounds are within the range of 6.3-21.5 μM, and they are quite similar to the activity of two best and most extensively investigated natural benzyl isothiocyanate (A) and phenethyl isothiocyanate (B). Preliminary studies utilizing the cell cycle and reduced glutathione level analysis performed on A549 lung cancer cell line using representative compounds revealed important differences in the mechanism of action possibly correlated with their chemical properties. Hydrophobic compounds react mainly with the cytosolic glutathione reduced leading to its depletion, causing an oxidative stress and cell cycle arrest in G0/G1 phase. On the other hand, hydrophilic compounds cause moderate cell cycle arrest and massive cell death associated with moderate reduced glutathione depletion. These suggest that significant changes in the chemical structure of isothiocyanates, which do not lead to the significant changes in antiproliferative activity, but simultaneously cause a differences in the mechanism of action are possible.     

Biological activity of pyrazole and imidazole-dehydroepiandrosterone derivatives on the activity of 17β-hydroxysteroid dehydrogenase

The enzyme type 5 17β-hydroxysteroid dehydrogenase 5 (17β-HSD5) catalyzes the transformation of androstenedione (4-dione) to testosterone (T) in the prostate. This metabolic pathway remains active in cancer patients receiving androgen deprivation therapy. Since physicians seek to develop advantageous and better new treatments to increase the average survival of these patients, we synthesized several different dehydroepiandrosterone derivatives. These compounds have a pyrazole or imidazole function at C-17 and an ester moiety at C-3 and were studied as inhibitors of 17β-HSD5. The kinetic parameters of this enzyme were determined for use in inhibition assays. Their pharmacological effect was also determined on gonadectomized hamsters treated with Δ⁴-androstenedione (4-dione) or testosterone (T) and/or the novel compounds. The results indicated that the incorporation of a heterocycle at C-17 induced strong 17β-HSD5 inhibition. These derivatives decreased flank organ diameter and prostate weight in castrated hamsters treated with T or 4-dione. Inhibition of 17β-HSD5 by these compounds could have therapeutic potential for the treatment of prostate cancer and benign prostatic hyperplasia.     

δ-Toxin,unlike melittin,has only hemolytic activity and no antimicrobial activity: Rationalization of this specific biological activity

The antimicrobial activity of a synthetic peptide corresponding to -hemolysin had been examined. The peptide didnot exhibit antimicrobial activity against gram negative and gram positive micro-organisms unlike other hemolytic peptides like melittin. This lack of antibacterial activity arises due to the inability of -hemolysin to perturb the negatively charged bacterial cell surface and permeabilize the bacterial plasma membrane. However, the red blood cell surface has a structure considerably different from bacteria, and does not act as a barrier to molecules reaching the lipid membrane. Hence -toxin can lyse erythrocytes. Thus, the specificity in biological activity has been rationalized in terms of differences, in the interaction of the toxin with the bacterial and red blood cell surfaces.     

Synthesis and structure–activity relationships of cassiarin A as potential antimalarials with vasorelaxant activity

Cassiarin A 1, a tricyclic alkaloid, isolated from the leaves of Cassia siamea (Leguminosae), shows powerful antimalarial activity against Plasmodium falciparum in vitro as well as P. berghei in vivo, which may be valuable leads for novel antimalarials. Interactions of parasitized red blood cells (pRBCs) with endothelium in aorta are especially important in the processes contribute to the pathogenesis of severe malaria. Nitric oxide (NO) reduces endothelial expression of receptors/adhesion molecules used by pRBC to adhere to vascular endothelium, and reduces cytoadherence of pRBC to vascular endothelium. Cassiarin A 1 showed vasorelaxation activity against rat aortic ring, which may be related with NO production. A series of a hydroxyl and a nitrogen-substituted derivatives and a dehydroxy derivative of 1 have been synthesized as having potent antimalarials against P. falciparum with vasodilator activity, which may reduce cytoadherence of pRBC to vascular endothelium. Cassiarin A 1 exhibited a potent antimalarial activity and a high selectivity index in vitro, suggesting that the presence of a hydroxyl and a nitrogen atom without any substituents may be important to show antimalarial activity. Relative to cassiarin A, a methoxy derivative showed more potent vasorelaxant activity, although it did not show improvement for inhibition of P. falciparum in vitro. These cassiarin derivatives may be promising candidates as antimalarials with different mode of actions.     

Physical activity and postprandial lipidemia: are energy expenditure and lipoprotein lipase activity the real modulators of the positive effect?

Historically, the link between elevated cholesterol and increased risk of cardiovascular disease has been based on fasting measurements. This is appropriate for total, low-density lipoprotein and high-density lipoprotein cholesterol. However, triglyceride concentrations vary considerably throughout the day in response to the regular consumption of food and drink. Recent findings indicate that postprandial triglyceride concentrations independently predict future cardiovascular risk. Potential modulators of postprandial lipidemia include meal composition and physical activity. Early cross sectional studies indicated that physically active individuals had a lower postprandial lipidemic response compared to inactive individuals. However, the effect of physical activity on postprandial lipidemia is an acute phenomenon, which dissipates within 60 h of a single bout of exercise. Total exercise induced energy expenditure, rather than duration or intensity of the physical activity is commonly reported to be a potent modulator of postprandial lipidemia. However, the pooled results of studies in this area suggest that energy expenditure exerts most of its influence on fasting triglyceride concentrations rather than on the incremental change in triglyceride concentrations seen following meal consumption. It seems more likely that energy expenditure is one component of a multifactorial list of mediators that may include local muscle contractile activity, and other yet to be elucidated mechanisms.     

Synthesis and structure–activity relationships of phenyl-substituted coumarins with anti-tubercular activity that target FadD32

In an effort to develop new and potent agents for therapy against tuberculosis, a high-throughput screen was performed against Mycobacterium tuberculosis strain H37Rv. Two 6-aryl-5,7-dimethyl-4-phenylcoumarin compounds 1a and 1b were found with modest activity. A series of coumarin derivatives were synthesized to improve potency and to investigate the structure–activity relationship of the series. Among them, compounds 1o and 2d showed improved activity with IC₉₀ of 2 μM and 0.5 μM, respectively. Further optimization provided compound 3b with better physiochemical properties with IC₉₀ 0.4 μM which had activity in a mouse model of infection. The role of the conformation of the 4- and 6-aryl substituents is also described.     

Synthesis and antibacterial activity of catecholate–ciprofloxacin conjugates

The development of an efficient route to obtain artificial siderophore–antibiotic conjugates active against Gram-negative bacteria is crucial. Herein, a practical access to triscatecholate enterobactin analogues linked to the ciprofloxacin along with their antibacterial evaluation are described. Two series of conjugates were obtained with and without a piperazine linker which is known to improve the pharmacokinetics profile of a drug. A monocatecholate–ciprofloxacin conjugate was also synthesized and evaluated. The antibacterial activities against Pseudomonas aeruginosa for some conjugates are related to the iron concentration in the culture medium and seem to depend on the bacterial iron uptake systems.     

Leucine enkephalin-tyrosinase reaction products — Identification and biological activity

Leucine enkephalin (1 mM) was reacted with mushroom tyrosinase under reductive conditions (ascorbic acid, 50 mM). Reaction products were isolated by high-performance liquid chromatography and identified using electrospray ionization mass spectrometry. The products of the reaction were found to be hydroxylated at the Tyr¹ moiety of the peptide. The major product was a monohydroxylated derivative of leucine enkephalin ([HO-Tyr¹]LE) and the minor product of the reaction was a dihydroxylated derivative ([(HO)₂-Tyr¹]LE). The affinity of [HO-Tyr¹]LE to receptors in rat brain homogenate was compared to that of leucine enkephalin itself. Hydroxylation of LE was found to decrease receptor affinity to both μ and δ opioid receptor sites by a factor of about 20.     

Synthesis and activity of α-glucosidase produced byBifidobacterium pseudolongum

Bifidobacterium pseudolongum NCFB 2244 grew on starch as sole source of carbon and energy, but cell yields and specific growth rates were considerably lower than on glucose (=0.19±0.04 and 0.38±0.09 respectively). Amylase activity was not detected in cultures of this bacterium, but cell-associated -glucosidase was constitutively produced. Analysis of -glucosidase activity from cell extracts by preparative isoelectric focusing gave two peaks of activity with apparent isoelectric points of 3.9 (Enzyme I) and 4.2 (Enzyme II), corresponding to threefold and fourfold purification factors respectively. No -glucosidase activity was detected with Enzyme I after gel-filtration chromatography on Sephadex G150. However, activity was recovered in samples containing Enzyme II, indicating the protein had a molecular mass of approximately 126 kDa. This was subsequently confirmed by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). These results show that the restricted ability ofB. pseudolongum to utilize starch as a carbohydrate source is owing to synthesis of at least one, and possibly two, -glucosidase(s).     

Physiological activity and receptor binding of 9α fluorohydrocortisone

Hepatic gluconeogenic stimulation by 9αfluorocortisol was associated with saturation of GR₁ and GR₃ entities of the glucocorticoid specific receptor (GR), even in presence of spironolactone; renal glycogen levels were not altered. Binding to MR₁ and MR₂ components of the mineralocorticoid specific receptor (MR) in the kidney persisted even in presence of 100 fold excess of nonradioactive corticosterone although this was totally abolished by cold equimolar spironolactone. These data suggest that this fluorinated derivative may be particularly appropriate in studying organ specific responses.