Nitro-substituted tetrahydroindolizines and homologs: Design,kinetics, and mechanism of α-glucosidase inhibition

A series of 1-[(methylsulfonyl)methyl]-2-nitro-5,6,7,8-tetrahydroindolizines and homologs were designed, prepared, and evaluated as non-sugar-type α-glucosidase inhibitors. The inhibitory activity appeared to be related to cyclo homologation with the best congeners being tetrahydroindolizines. The introduction of a methoxycarbonyl group as an additional hydrogen bond acceptor into the exocyclic methylene group was beneficial affording the most potent congener 3e (half maximal inhibitory concentration, IC₅₀ = 8.0 ± 0.1 μM) which displayed 25-fold higher inhibitory activity than 1-deoxynojirimycin (2, IC₅₀ = 203 ± 9 μM)—the reference compound. Kinetic analysis indicated that compound 3e is a mixed inhibitor with preference for the free enzyme over the α-glucosidase–substrate complex (K_i,free = 3.6 μM; K_i,bound = 7.6 μM). Molecular docking experiments were in agreement with kinetic results indicating reliable interactions with both the catalytic cleft and other sites. Circular dichroism spectroscopy studies suggested that the inhibition exerted by 3e may involve changes in the secondary structure of the enzyme. Considering the relatively low molecular weight of 3e together with its high fraction of sp³ hybridized carbon atoms, this nitro-substituted tetrahydroindolizine may be considered as a good starting point towards new leads in the area of α-glucosidase inhibitors.     

Toxicity of Amyloid β Peptide: Tales of Calcium,Mitochondria, and Oxidative Stress

Alzheimer's disease (AD) is characterized by the accumulation of amyloid-beta (Abeta) peptides. Although the disease undoubtedly reflects the interaction of complex multifactorial processes, Abeta itself is toxic to neurons in vitro and the load of Abeta in vivo correlates well with the degree of cognitive impairment. There has therefore been considerable interest in the mechanism(s) of Abeta neurotoxicity. We here review the basic biology of Abeta processing and consider some of the major areas of focus of this research. It is clear that both AD and Abeta toxicity are characterized by oxidative stress, alterations in the activity of enzymes of intermediary metabolism, and mitochondrial dysfunction, especially impaired activity of cytochrome c oxidase. Studies in vitro also show alterations in cellular calcium signaling. We consider the mechanisms proposed to mediate cell injury and explore evidence to indicate which of these many changes in function are primary and which secondary.     

Determination of the transgalactosylation activity of Aspergillus oryzae β-galactosidase: effect of pH, temperature, and galactose and glucose concentrations

The catalytic potential of β-galactosidase is usually determined by its hydrolytic activity over natural or synthetic substrates. However, this method poorly predicts enzyme behavior when transglycosylation instead of hydrolysis is being performed. A system for determining the transgalactosylation activity of β-galactosidase from Aspergillus oryzae was developed, and its activity was determined under conditions for the synthesis of galacto-oligosaccharides and lactulose. Transgalactosylation activity increased with temperature up to 55 °C while the effect of pH was mild in the range from pH 2.5 to 5.5, decreasing at higher values. The effect of glucose and galactose on transgalactosylation activity was also assessed both in the reactions for the synthesis of galacto-oligosaccharides and lactulose and also in the reaction of hydrolysis of o-nitrophenyl β-d-galactopiranoside. Galactose was a competitive inhibitor and its effect was stronger in the reactions of transgalactosylation than in the reaction of hydrolysis. Glucose was a mild activator of β-galactosidase in the reaction of hydrolysis, but its mechanism of action was more complex in the reactions of transgalactosylation, having this positive effect only at low concentrations while acting as an inhibitor at high concentrations. This information is relevant to properly assess the effect of monosaccharides during the reactions of the synthesis of lactose-derived oligosaccharides, such as galacto-oligosaccharides and lactulose.     

Collagen modulates cell shape and cytoskeleton of embryonic corneal and fibroma fibroblasts: Distribution of actin, α-actinin,and myosin

In the embryo, fibroblasts migrating through extracellular matrices (ECM) are generally elongate in shape, exhibiting a leading pseudopodium with filopodial extensions, and a trailing cell process. Little is known about the mechanism of movement of embryonic cells in ECM, for studies of fibroblast locomotion in the past have been largely confined to observations of flattened cells grown on planar substrata. We confirm here that embryonic avian corneal fibroblasts migrating within hydrated collagen gels in vitro have the bipolar morphology of fibroblasts in vivo, and we show for the first time that highly flattened gerbil fibroma fibroblasts, grown as cell lines on planar substrata, can also respond to hydrated collagen gels by becoming elongate in shape. We demonstrate that the collagen-mediated change in cell shape is accompanied by dramatic rearrangement of the actin, α-actinin, and myosin components of the cytoskeleton. By immunofluorescence, the stress fibers of the flattened corneal fibroblasts grown on glass are seen to stain with antiactin, anti-α-actinin, and antimyosin, as has been reported for fibroma and other fibroblasts grown on glass. Stress fibers, adhesion plaques, and ruffles do not develop when the corneal or fibroma fibroblast is grown in ECM; these features seem to be a response to strong attachment of the cell underside to a planar substratum. When the fibroblasts are grown in ECM, antimyosin staining is distributed diffusely through the cytoplasm. Antiactin and anti-α-actinin stain the microfilamentous cell cortex strongly. We suggest that locomotion of the fibroblast in ECM is accompanied by adhesion of the cell to the collagen fibrils and may involve an interaction of the myosin-rich cytosol with the actin-rich filamentous cell cortex. Interestingly, the numerous filopodia that characterize the tips of motile pseudopodia of cells in ECM are very rich in actin and α-actinin, but seem to lack myosin; if filopodia use myosin to move, the interaction must be at a distance. Soluble collagen does not convert flattened fibroblasts on planar substrata to bipolar cells. Thus, the effect of collagen on the fibroblast cytoskeleton seems to depend on the presence of collagen fibrils in a gel surrounding the cell.     

Rates of entry and oxidation of acetate,glucose, d(?)-β-hydroxybutyrate,palmitate, oleate and stearate,and rates of production and oxidation of propionate and butyrate in fed and starved sheep

1. Rates of entry and oxidation of a range of metabolites have been measured in tracheostomized sheep (diet, 800g. of lucerne chaff and 100g. of maize/day) by combining isotope-dilution techniques with the continuous measurement of total respiratory gas exchange, and ¹⁴CO₂ production during the intravenous or intraruminal infusion of ¹⁴C-labelled substrates. 2. Mean entry rates in fed and starved (24hr.) sheep respectively, expressed as mg./min./kg. body wt.^0·75, were: glucose, 5·0 (range 4·8–5·1, 2 observations) and 3·8 (3·2–4·2, 4); acetate, 10·8 (9·1–13·5, 4) and 5·8 (1); d(−)-β-hydroxybutyrate, 1·4 (1) and 1·5 (0·8–2·4, 4); palmitate, oleate and stearate (starved sheep only) 1·0 (0·6–1·9, 7), 0·9 (0·2–1·6, 10) and 0·9 (0·5–1·1, 11) respectively. 3. Production rates of propionate and butyrate in continuously feeding sheep were 6·4 (4·7–8·3, 4) and 4·3 (3·4–6·1, 4) mg./min./kg.^0·75 respectively, and in starved (24hr.) sheep were 2·5 (2·2–2·9, 2) and 1·0 (0·8–1·2, 2) mg./min./kg.^0·75 respectively. 4. Calculated terminal values for the specific radioactivity of respiratory ¹⁴CO₂ during measurements of entry rates and production rates were used to calculate the contributions of individual substrates to overall oxidative metabolism. Mean values for fed and starved sheep respectively were: glucose, 9·1 (8·6–9·6, 2) and 11·2 (5·9–15·1, 4)%; acetate, 31·6 (26·8–38·1, 4) and 22·1 (1)%; d(−)-β-hydroxybutyrate, 10·4 (1) and 4·8 (1·9–7·7, 4)%; propionate, 23·0 (13·8–29·9, 4) and 7·1 (6·8–7·4, 2)%; butyrate, 16·5 (13·7–20·5, 4) and 5·3 (5·2–5·3, 2)%; palmitate, oleate and stearate (starved sheep only), 4·7 (2·0–7·7, 7), 4·0 (1·2–6·6, 10) and 4·4 (3·8–5·8, 9)% respectively. The sum of these values for individual substrates in fed and starved sheep, excluding that of β-hydroxybutyrate and after correction of the glucose value for the known interrelations of this substrate with propionate, accounted for 76% and 58% respectively of total production of carbon dioxide. 5. Calculations based on the proportion of substrate entry directly oxidized indicated that the substrates studied accounted for 63% (fed sheep) and 43% (starved sheep) of total energy expenditure measured by oxygen uptake. The contribution of β-hydroxybutyrate was excluded, and corrections were made for glucose–propionate interrelations, and for the different rates of oxidation of the methyl and carboxyl fragments of acetate. 6. The present results have been combined with those obtained earlier in this Laboratory to examine the relationships between rates of substrate entry and oxidation, and concentrations of substrate in blood. Rates of entry of acetate, glucose, d(−)-β-hydroxybutyrate, palmitate and oleate (but not stearate) were well correlated with concentration in blood, and substrate contribution to production of carbon dioxide showed a similar correlation to blood concentration, except with glucose. 7. It was concluded that the general technique is of potential value in providing valid quantitative parameters of animal metabolism.     

The Evolution of the idea of homeostasis: Determinism,stochastics, and chaos–self-organization

Homeostasis is a basic concept in investigating all living systems. Homeostasis and life are synonyms to a certain extent. The concept of homeostasis has been studied and developed for more than 150 years; however, only the 21st century brought us close to understanding homeostasis as a state of a biosystem and a continuous process. The chaos and self-organization theory (CSOT) proves that the conventional views based on determinism (functional analysis) or stochastics (when stochastic uncertainty or certainty occurs) cannot describe homeostasis as defined by W.B. Cannon and his followers. The new CSOT shows a clear boundary between determinism, stochastics (and deterministic chaos), and third-type systems, which have five special properties (principles of self-organization) and can be described in terms of quasiattractors. Kinematics is understood as the motion of quasi-attractors in the phase spaces of states. Complexity is introduced as the rate of evolution of complex biological systems, differing from the definition by Prigogine–Glansdorff. At the same time, concepts of uncertainty of the first and second types and an analog of the Heisenberg calculus are introduced for complexity. According to these concepts, complex biosystems are transferred from the area of traditional science to a new area of the theory of chaos and self-organization.     

Termination of translation: interplay of mRNA,rRNAs and release factors?

Termination of translation in eukaryotes has focused recently on functional anatomy of polypeptide chain release factor, eRF1, by using a variety of different approaches. The tight correlation between the domain structure and different functions of eRF1 has been revealed. Independently, the role of prokaryotic RF1/2 in GTPase activity of RF3 has been deciphered, as well as RF3 function itself.     

Adsorption of glycinin and β-conglycinin on silica and cellulose: surface interactions as a function of denaturation, pH, and electrolytes

Soybean proteins have found uses in different nonfood applications due to their interesting properties. We report on the kinetics and extent of adsorption on silica and cellulose surfaces of glycinin and β-conglycinin, the main proteins present in soy. Quartz crystal microgravimetry (QCM) experiments indicate that soy protein adsorption is strongly affected by changes in the physicochemical environment. The affinity of glycinin and the mass adsorbed on silica and cellulose increases (by ca. 13 and 89%, respectively) with solution ionic strength (as it increases from 0 to 100 mM NaCl) due to screening of electrostatic interactions. In contrast, β-conglycinin adsorbs on the same substrates to a lower extent and the addition of electrolyte reduces adsorption (by 25 and 57%, respectively). The addition of 10 mM 2-mercaptoethanol, a denaturing agent, reduces the adsorption of both proteins with a significant effect for glycinin. This observation is explained by the cleavage of disulfide bonds which allows unfolding of the molecules and promotes dissociation into subunits that favors more compact adsorbed layer structures. In addition, adsorption of glycinin onto cellulose decreases with lowering the pH from neutral to pH 3 due to dissociation of the macromolecules, resulting in flatter adsorbed layers. The respective adsorption isotherms fit a Langmuir model and QCM shifts in energy dissipation and frequency reveal multiple-step kinetic processes indicative of changes in adlayer structure.     

Discovery of α-amidosulfones as potent and selective agonists of CB2: Synthesis,SAR, and pharmacokinetic properties

A series of α-amidosulfones were found to be potent and selective agonists of CB₂. The discovery, synthesis, and structure–activity relationships of this series of agonists are reported. In addition, the pharmacokinetic properties of the most promising compounds are profiled.     

Adolescents’ Use of Care for Behavioral and Emotional Problems: Types,Trends, and Determinants

Objective

While adolescents use various types of care for behavioral and emotional problems, evidence on age trends and determinants per type is scarce. We aimed to assess use of care by adolescents because of behavioral and emotional problems, overall and by type, and its determinants, for ages 10–19 years.

Methods

We obtained longitudinal data on 2,230 adolescents during ages 10–19 from four measurements regarding use of general care and specialized care (youth social care and mental healthcare) in the preceding 6 months, the Child Behavior Checklist (CBCL) and Youth Self-Report, and child and family characteristics. We analyzed data by multilevel logistic regression.

Results

Overall rates of use increased from 20.1% at age 10/11 to 32.2% at age 19: general care was used most. At age 10/11 use was higher among boys, at age 19 among girls. Use of general care increased for both genders, whereas use of specialized care increased among girls but decreased among boys. This differential change was associated with CBCL externalizing and internalizing problems, school problems, family socioeconomic status, and parental divorce. Preceding CBCL problems predicted more use: most for mental health care and least for general care. Moreover, general care was used more frequently by low and medium socioeconomic status families, with odds ratios (95%-confidence intervals): 1.52 (1.23;1.88) and 1.40 (1.17;1.67); youth social care in case of parental divorce, 2.07 (1.36;3.17); and of special education, 2.66 (1.78;3.95); and mental healthcare in case of special education, 2.66 (1.60;4.51).

Discussion

Adolescents with behavioral and emotional problems use general care most frequently. Overall use increases with age. Determinants of use vary per type.     

Methodology and Problems of Protein‐Ligand Docking: Case Study of Dihydroorotate Dehydrogenase,Thymidine Kinase,and Phosphodiesterase 4

Abstract

The docking methodology was applied to three different therapeutically interesting enzymes: human dihydroorotate dehydrogenase (DHODH), Herpes simplex virus type I thymidine kinase (HSV1 TK) and human phosphodiesterase 4 (PDE4). Programs FlexX, AutoDock and DOCK where used. The three targets represent three distinct cases. For DHODH and HSV1 TK, the binding modes of substrate and inhibitors within the active site are known, while the binding orientation of cAMP within PDE4 has been solely hypothesized. Active site of DHODH is mainly hydrophobic and the binding mode of the inhibitor brequinar was used as a template for evaluating the docking strategies. The presence of cofactors revealed to be crucial for the definition of the docking site. The HSV1 TK active site is small and polar and contains crystal water molecules and ATP. Docking of thymidine and aciclovir (ACV) within the active site was analyzed by keeping or removing water molecules. It showed the crucial role of water in predicting the binding of pyrimidines and purines. The crystal structure of PDE4 contains magnesium and zinc cations as well as catalytic water molecule but no ligand. Several docking experiments of cAMP and rolipram were performed and the results showed clear‐cut dependence between the ligand orientation and the presence of metals in the active site. All three cases show specific problems of the docking methodology, depending on the character of the active site.     

Reproductive and socioeconomic determinants of child survival: Confounded,interactive, and age‐dependent effects

Abstract

Studies of infant and child mortality have evolved to distinguish between two sets of explanatory variables—factors related to reproductive or maternal characteristics and socioeconomic factors, generally described as characteristics of the family or household. Almost all multivariate analyses include variables from each of these two sets, but there has been little consideration of the relationship between them. We examine how these two sets of variables jointly affect mortality. We test first for confounded effects by examining socioeconomic effects while excluding and then including reproductive variables in nested multivariate models. Next, we look for age‐dependent effects among the explanatory variables and find that reproductive and socioeconomic factors affect mortality at differing ages of children. Finally, we examine interactive effects of the two sets of variables. We conclude that the higher mortality observed among the low status groups is not a result of greater concentration of poor reproductive patterns in those groups. Instead, higher status groups probably have more resources available for combating the negative effects of the same high‐risk reproductive patterns.     

Diagnosis of “Rheumatoid Variants”: Ankylosing Spondylitis,the Arthritides of Gastrointestinal Diseases and Psoriasis,and Reiter's Syndrome

Four rheumatic diseases—ankylosing spondylitis, the arthritis accompanying ulcerative colitis or regional enteritis, psoriatic arthropathy, and Reiter''s syndrome—formerly considered to be forms of rheumatoid arthritis, are now distinguished from that disorder and should be recognized by the physician as entities. These arthritides may be distinguished from each other by a number of clinical and radiographic characteristics, principally (1) the roentgenographic appearance of the spine when spondylitis is present, (2) the location of periosteal new bone formation, (3) the location of arthritis in the joints of the limbs, and (4) the presence of characteristic skin lesions.     

Novel conjugates of podophyllotoxin and coumarin: Synthesis,cytotoxicities, cell cycle arrest,binding CT DNA and inhibition of Topo IIβ

A series of conjugates of podophyllotoxin and coumarin were prepared using the click reaction, and their cytotoxicities against A549, HepG2, HeLa, and LoVo cells were evaluated. Among them, compound 14e exhibited the strongest cytotoxicities against these cancer cells with IC₅₀ values of 4.9–17.5 μM. Furthermore, 14e disrupted microtubules and induced cell cycle arrest at G1 phase by regulating P21 and Cyclin D1 in LoVo cells. In addition, 14e bond CT DNA and selectively inhibited Topo IIβ over Topo IIα. Molecular docking model showed that 14e appeared to form stable hydrogen bonds with several DNA bases and residue Gln778. Taken together, these conjugates have the potential to be developed as anti-tumor drugs.     

Functions of Aβ, sAPPα and sAPPβ : similarities and differences

Amyloid peptide (Aβ) is derived from the cleavage of amyloid precursor protein (APP), which also generates the soluble peptide APPβ (sAPPβ). An antagonist and major APP metabolic pathway involves cleavage by alpha secretase, which releases sAPPα. Although soluble Aβ oligomers are neurotoxic, Aβ monomers share similar properties with sAPPα. These include neurotrophic and neuroprotective effects, as well as stimulation of neural-progenitor proliferation. The properties of Aβ monomers and the neurotrophic capacity of sAPPβ to stimulate axonal outgrowth suggest that Aβ production is not deleterious per se. Consequently, therapeutic strategies for Alzheimer's disease that are targeted at Aβ-cleaving enzymes should modulate rather than inhibit Aβ generation. These strategies should focus on the factors that induce the conversion of Aβ monomers into toxic soluble oligomers. Another interesting therapeutic approach is to focus on the mechanisms of the different properties of sAPPα. Indeed, increasing sAPPα levels could shift proliferating cells towards tumorigenesis. In contrast to its neuroprotective effects, sAPPα is also able to activate microglia, leading to neurotoxicity. Understanding the mechanisms that underlie the different properties of sAPPα could therefore lead to the development of therapeutic strategies against Alzheimer's disease, which could be curative as well as preventive.     

Serum γ-glutamyltransferase: independent predictor of risk of diabetes, hypertension, metabolic syndrome, and coronary disease

Serum γ-glutamyltransferase (GGT) is associated with oxidative stress and hepatic steatosis. The extent to which its value in determining incident cardiometabolic risk (coronary heart disease (CHD), metabolic syndrome (MetS), hypertension and type 2 diabetes) is independent of obesity needs to be further explored in ethnicities. After appropriate exclusions, a cohort of 1,667 adults of a general population (age 52 ±11 years) was evaluated prospectively at 4 year's follow-up using partly Cox proportional hazard regressions. GGT activity was measured kinetically, and values were log-transformed for analyses. MetS was identified by Adult Treatment Panel-III criteria modified for male abdominal obesity. Median (interquartile range) GGT activity was 24.9 (17.0; 35.05) U/l in men, 17.0 (12.3; 24.0) U/l in women. In linear regression analysis, while smoking status was not associated, (male) sex, sex-dependent age, alcohol usage, BMI, fasting triglycerides and C-reactive protein (CRP) were significant independent determinants of circulating GGT. Each 1-s.d. increment in (= 0.53 ln GGT) GGT activity significantly predicted in each sex incident hypertension (hazard ratio (HR) 1.20 (95% confidence interval (CI) 1.10; 1.31)), and similarly MetS, after adjustment for age, alcohol usage, smoking status, BMI and menopause. Strongest independent association existed with diabetes (HR 1.3 (95% CI 1.1; 1.5)) whereas GGT activity tended to marginally predict CHD independent of total bilirubin but not of BMI. Higher serum total bilirubin levels were protective against CHD risk in women. We conclude that elevated serum GGT confers, additively to BMI, risk of hypertension, MetS, and type 2 diabetes but only mediates adiposity against CHD risk.     

On the nature of facultative and constitutive CAM: environmental and developmental control of CAM expression during early growth of Clusia, Kalanchöe, and Opuntia

The capacity to induce crassulacean acid metabolism developmentally (constitutive CAM) and to up-regulate CAM expression in response to drought stress (facultative CAM) was studied in whole shoots of seven species by measuring net CO(2) gas exchange for up to 120 day-night cycles during early growth. In Clusia rosea, CAM was largely induced developmentally. Well-watered seedlings began their life cycle as C(3) plants and developed net dark CO(2) fixation indicative of CAM after the initiation of the fourth leaf pair following the cotyledons. Thereafter, CAM activity increased progressively and drought stress led to only small additional, reversible increases in dark CO(2) fixation. In contrast, CAM expression was overwhelmingly under environmental control in seedlings and mature plants of Clusia pratensis. C(3)-type CO(2) exchange was maintained under well-watered conditions, but upon drought stress, CO(2) exchange shifted, in a fully reversible manner, to a CAM-type pattern. Clusia minor showed CO(2) exchange reponses intermediate to those of C. rosea and C. pratensis. Clusia cretosa operated in the C(3) mode at all times. Notably, reversible stress-induced increases of dark CO(2) fixation were also observed during the developmental progression to pronounced CAM in young Kalancho? daigremontiana and Kalancho? pinnata, two species considered constitutive CAM species. Drought-induced up-regulation of CAM was even detected in young cladodes of a cactus, Opuntia ficus-indica, an archetypal constitutive CAM species. Evidently, the defining characteristics of constitutive and facultative CAM are shared, to variable degrees, by all CAM species.