Synthesis of an insulin analogue embodying a strongly fluorescent moiety, [19-Tryptophan-A]insulin

As part of our aim to study the conformation of insulin in solution by time-resolved fluorescence spectroscopy, we have synthesized the analogue [19-Tryptophan-A]insulin. In this compound, the tyrosine residue at position 19 of the A-chain of insulin, one of the most strongly conserved residues in insulins from various species, is substituted with the strongly fluorescent tryptophan residue. [19-Tryptophan-A]insulin displays 4.1±1.9% of the potency of natural insulin in binding to the insulin receptor from rat liver plasma membranes, 5.0±2.3% in stimulating lipogenesis in rat adipocytes, and 75.7±4% of the potency of insulin in radioimmunoassay. In connection with our previous work, these data indicate that an aromatic side chain at position A¹⁹ of insulin seems necessary but not sufficient for high biological activity. We further conclude that in regard to the immunogenic determinants of insulin, tryptophan in position A¹⁹ is an essentially neutral substitution for tyrosine in that position, in sharp contrast to the situation with regard to biological activity.     

In vitro binding to and in vivo effect on the cytosol and nuclear progesterone receptors of various progestins,and their relationship to synthesis of uteroglobin in rabbit uterus

In vitro binding affinities of various progestins to cytosol and nuclear progesterone receptors of rabbit uterus were determined and correlated with the biological potency of these steroids. In addition, cytosol and nuclear progesterone receptor levels were measured after a 5-day administration of different progestins (0.5 mg/kg daily) with variable biologic activites. The receptor levels were compared with the bilological response; the induction of uteroglobin synthesis. Cytosol and nuclear progesterone receptors had identical steroid binding properties (r = 0.98). The correlation between the in vitro binding affinity (cytosol or nuclear) and the in vivo biologic activity of the steroids was good (r = 0.73). After a 5-day treatment with progestins, the nuclear receptor concentration correlated in an inverse manner (r = ?0.84) with the uterine fluid unteroglobin concentration. A similar, but slightly weaker correlation (r = ?0.81) was also found for the cytosol receptor content and uteroglobin secretion. These data indicate that not only nuclear, but also cytosol progesterone receptor levels decrease in the rabbit uterus during chronic hormone action. Decline in the nuclear progesterone receptor content seemed to occur during treatment with all progestational steroids, while onlyi progestins with high biological potency were capable of decreasing the cytosol receptor content.     

Antibacterial activity of Turkish propolis and its qualitative and quantitative chemical composition

The antibacterial activity of propolis from different regions of Turkey was studied, accompanied by TLC and GC-MS analyses of its chemical composition and spectrophotometric quantification of the most important active principles. All six samples were active against the bacterial test strains used; however, samples 1 (Yozgat), 2 (Izmir) and 3 (Kayseri) were more active than samples 4 (Adana), 5 (Erzurum) and 6 (Artvin). By TLC comparison all samples were found to contain poplar taxonomic markers but in samples 4 (Adana), 5 (Erzurum) and 6 (Artvin), different substances were observed, which were not present in P. nigra L. bud exudate. The typical poplar samples 1 (Yozgat), 2 (Izmir) and 3 (Kayseri) displayed very similar phenolic and flavonoid content. Samples 4 (Adana), 5 (Erzurum) and 6 (Artvin) were characterized by low phenolic and very low flavonoid concentrations. Qualitative analysis by GC-MS revealed that sample 4 (Adana) contained diterpenic acids and high percent of cinnamyl cinnamate, sample 5 (Erzurum)-significant amounts of hydroxy fatty acids and triterpenic alcohoLs, and sample 6 (Artvin)-phenolic glycerides, characteristic for the bud exudate of Populus euphratica Oliv. The results confirm the importance of phenolics for propolis antibacterial activity, and the significance of P. nigra L. as a propolis source, which provides the hive with the best defense against microorganisms.     

Modeling of substrate and inhibitor binding to phospholipase A2.

Molecular graphics and molecular mechanics techniques have been used to study the mode of ligand binding and mechanism of action of the enzyme phospholipase A2. A substrate-enzyme complex was constructed based on the crystal structure of the apoenzyme. The complex was minimized to relieve initial strain, and the structural and energetic features of the resultant complex analyzed in detail, at the molecular and residue level. The minimized complex was then used as a basis for examining the action of the enzyme on modified substrates, binding of inhibitors to the enzyme, and possible reaction intermediate complexes. The model is compatible with the suggested mechanism of hydrolysis and with experimental data about stereoselectivity, efficiency of hydrolysis of modified substrates, and inhibitor potency. In conclusion, the model can be used as a tool in evaluating new ligands as possible substrates and in the rational design of inhibitors, for the therapeutic treatment of diseases such as rheumatoid arthritis, atherosclerosis, and asthma.     

Cation binding effects on the pH, thermal and urea denaturation transitions in alpha-lactalbumin

The binding of monovalent (Na+, K+) and divalent (Ca2+, Mg2+) cations to bovine alpha-lactalbumin at 20 and 37 degrees C has been studied by means of intrinsic protein fluorescence. The values of apparent binding constants for these ions obtained at 37 degrees C are about one order of magnitude lower than those measured at 20 degrees C. Urea and alkali (pH greater than 10) induce unfolding transitions which involve stable partially unfolded intermediates for all metal ion-bound forms of alpha-lactalbumin. Heating induces similar partially unfolded states. Nevertheless, the partially unfolded states induced by heating, urea, alkaline or acidic treatments are somewhat different in their tryptophan residue environment properties. The results have been interpreted in terms of a simple scheme of equilibria between metal-free and metal-bound forms in their native, partially unfolded and unfolded states. The scheme provides an approach to the quantitative interpretation of any transition equilibrium shift induced by a low molecular mass species able to be bound by a protein.     

Rapid reprogramming of haemoglobin structure‐function exposes multiple dual‐antimicrobial potencies

The intrinsic cytotoxicity of cell‐free haemoglobin (Hb) has hampered the development of reliable Hb‐based blood substitutes for over seven decades. Notably, recent evidence shows that the Hb deploys this cytotoxic attack against invading microbes, albeit, through an unknown mechanism. Here, we unraveled a rapid molecular reprogramming of the Hb structure‐function triggered by virulent haemolytic pathogens that feed on the haem‐iron. On direct contact with the microbe, the Hb unveils its latent antimicrobial potency, where multiple antimicrobial fragments are released, each harbouring coordinated ‘dual‐action centres’: microbe binding and pseudoperoxidase (POX) cycle activity. The activated Hb fragments anchor onto the microbe while the juxtaposed POX instantly unleashes a localized oxidative shock, killing the pathogen‐in‐proximity. This concurrent action conceivably restricts the diffusion of free radicals. Furthermore, the host astutely protects itself from self‐cytotoxicity by simultaneously releasing endogenous antioxidants. We found that this decryption mechanism of antimicrobial potency is conserved in the ancient invertebrate respiratory protein, indicating its fundamental significance. Our definition of dual‐antimicrobial centres in the Hb provides vital clues for designing a safer Hb‐based oxygen carrier blood substitute.     

Opposite Contributions of Trunk and Leg Fat Mass with Plasma Lipase Activities: The Hoorn Study

Objective: Lipoprotein lipase (LPL) and hepatic lipase (HL) are essential in hydrolysis of triglyceride‐rich lipoproteins. LPL activity is negatively, whereas HL activity is positively, associated with total body fat. We determined the associations of trunk and leg fat mass with plasma LPL and HL activities in a cross‐sectional study. Research Methods and Procedures: LPL and HL activities were determined in post‐heparin plasma in a sample of 197 men and 209 women, 60 to 87 years of age. A total body DXA scan was performed to determine trunk and leg fat mass. Results: In women, but not in men, trunk fat mass was negatively associated with LPL activity, whereas leg fat mass was positively associated, after mutual adjustment and adjustment for age. Standardized βs (95% confidence interval) for trunk and leg fat mass were ?0.24 (?0.41; ?0.08) and 0.14 (?0.02; 0.31), respectively (interaction by sex, p = 0.03). Larger trunk fat mass was associated with higher HL activity in men [0.48 (0.28; 0.68)] and women [0.40 (0.24; 0.56)]. A negative association of leg fat mass and HL activity was observed in men, although not statistically significant [?0.13 (?0.33; 0.06)], and in women [?0.28 (?0.38; ?0.18)]. Discussion: Abdominal fat is associated with unfavorable and femoral fat with favorable LPL and HL activities in plasma.     

The role of substrate feeding earthworms (Lumbricidae) for bioturbation in a beechwood soil

Summary Burrow formation (burrow length and faeces production) of the substrate feeding earthworm Aporrectodea caliginosa (Savigny) in relation to three temperatures (5, 10 and 15°C) and soil moisture contents (48, 60 and 73% water of dry wt) was determined. Soil moisture content affected the burrowing activity of A. caliginosa only below a distinct threshold (60% water of dry wt). At sufficient moisture an, increase in temperature by 5° C approximately doubled the amount of egesta produced. Another substrate feeding earthworm (Octolasion lacteum (Örley)) showed a similar pattern of burrowing activity.The data for the dependence of the faeces production of A. caliginosa on soil temperature and moisture content were combined with field data. The amount of faeces produced by the population of this earthworm species in a beechwood on limestone was calculated to be about 4.23 kg ha^-1 a^-1. The amount of egesta produced by all substrate feeding species is assumed to exceed 6 kg ha^-1 a^-1, which is equivalent to a soil layer of approximately 9 mm. This group of earthworms is therefore considered to be of greater importance for bioturbation and the formation of mull than previously assumed.     

Enzyme activities in a clay-loam soil amended with various crop residues

Summary Amylase, dehydrogenase, arylsulphatase and phosphatases activities were measured in a clay-loam soil amended with seven different crop residues. All enzyme activities, except phosphomonoesterase, were generally higher in the derived soil samples than in the original soil. Addition of tobacco and sunflower residues caused an increase on most of the enzyme activities while tomato residues increased only the amylase and phosphodiesterase activities. As the enzyme activities were positively correlated to each other, a common source of the enzymes is suggested even though the coefficients of correlation demonstrate that only a low percentage of the variability can be ascribed to the interactions among enzyme activities.     

Sensitivity of progesterone receptors to aurintricarboxylic acid: Inhibition of nuclear uptake,ATP and DNA binding

When hen oviduct cytosol samples containing progesterone receptor complexed to [³H]progesterone were included with isolated nuclei in presence of 0.2 mM aurintricarboxylic acid, more than 50% inhibition occurred in the uptake of progesterone receptor by the nuclei. The activated form of progesterone receptor appeared to be more sensitive to the presence of aurintricarboxylic acid since pretreatment of non-activated progesterone receptor with the inhibitor and the subsequent removal of the latter prior to activation did not result in the inhibition of receptor uptake by the nuclei. Also, the binding of progesterone receptor to columns of DNA-cellulose or ATP-Sepharose was abolished under simmilar conditions. When nuclei, ATP-Sepharose or DNA-cellulose were preincubated with the inhibitor prior to the addition of receptor preparations, no such inhibition resulted indicating that the inhibitor may be interacting with the receptor protein and not complexing to ATP, DNA or sites in the nuclei. The steroid binding properties of progesterone receptor, however, remained intact under these conditions. Both A and B forms of progesterone receptor are equally sensitive to aurintricarboxylic acid presence when tested for their nuclear uptake. Aurintricarboxylic acid was also found to be very effective at low concentrations (0.25 mM) in eluting the receptor complexes off ATP-Sepharose columns without disrupting the steroid binding properties of progesterone receptor. Our results suggest that auintricarboxylic acid is an effective inhibitor of progesterone receptor and that it may be acting by interfering with a site(s) on progesterone receptor which may be exposed upon activation and are involved in such processes as ATP binding, nuclear uptake and DNA binding. These observations suggest the use of aurintricarboxylic acid as a chemical probe for the analysis of progesterone receptor.