Design and discovery of 1,3-benzodiazepines as novel dopamine antagonists

A series of novel 1,3-benzodiazapine based D1 antagonists was designed according to the understanding of pharmacophore models derived from SCH 23390 (1b), a potent and selective D1 antagonist. The new design features an achiral cyclic-amidine that maintains desired basicity. Solid phase synthesis was developed for SAR development of the novel dopamine antagonists.     

Synthesis and structure–activity relationships of N-substituted spiropiperidines as nociceptin receptor ligands: Part 2

A series of N-8 substituted analogs based upon the spiropiperidine core of the original lead compound 1 was synthesized. This lead has been elaborated to compounds to give compounds 2 and 3 (R = H) that exhibited high NOP binding affinity as well as selectivity against other known opioid receptors. These two series have been further functionalized at the amido nitrogen. The synthesis and structure–activity relationship (SAR) of these and related compounds are discussed.     

Causes of shear sensitivity of the toxic dinoflagellate Protoceratium reticulatum

Dinoflagellates have proven extremely difficult to culture because they are inhibited by low‐level shear forces. Specific growth rate of the toxic dinoflagellate Protoceratium reticulatum was greatly decreased compared with static control culture by intermittent exposure to a turbulent hydrodynamic environment with a bulk average shear rate that was as low as 0.3 s^?1. Hydrodynamic forces appeared to induce the production of reactive oxygen species (ROS) within the cells and this caused peroxidation of cellular lipids and ultimately cell damage. Exposure to damaging levels of shear rate correlated with the elevated level of lipoperoxides in the cells, but ROS levels measured directly by flow cytometry did not correlate with shear induced cell damage. This was apparently because the measured level of ROS could not distinguish between the ROS that are normally generated by photosynthesis and the additional ROS produced as a consequence of hydrodynamic shear forces. Continuously subjecting the cells to a bulk average shear rate value of about 0.3 s^?1 for 24‐h caused an elevation in the levels of chlorophyll a, peridinin and dinoxanthin, as the cells apparently attempted to counter the damaging effects of shear fields by producing pigments that are potential antioxidants. In static culture, limitation of carbon dioxide produced a small but measureable increase in ROS. The addition of ascorbic acid (0.1 mM) to the culture medium resulted in a significant protective effect on lipid peroxidation, allowing cells to grow under damaging levels of shear rates. This confirmed the use of antioxidant additives as an efficient strategy to counter the damaging effects of turbulence in photobioreactors where shear sensitive dinoflagellates are cultivated. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Apoptosis in fresh and cryopreserved buffalo sperm

The objectives of present study were (a) validation of annexin V/PI assay for estimation of sperm apoptosis in buffalo (Experiment 1) and (b) determining the effect of stages of cryopreservation on sperm apoptosis and its correlation with sperm motility and plasma membrane integrity (Experiment 2). In Experiment 1, different levels of apoptosis were artificially induced in buffalo semen (100 × 10⁶ sperm/aliquot) through graded doses of camptothecin (5, 10 and 20 μM/aliquot). Higher concentrations of camptothecin (10 and 20 μM) successfully (P < 0.05) induced apoptosis as compared to the lower (5 μM) dose and/or control. In Experiment 2, semen samples (n = 9, three pooled semen samples from each of the three buffalo bulls separately) were cryopreserved using vapor freezing. The mean percentage of apoptotic, necrotic and viable sperm did not differ between fresh and before freezing stages. However, freezing and thawing increased (P < 0.05) the percentage of apoptotic sperm (25.4 ± 0.6 vs. 36.5 ± 1.9) while decreased (P < 0.05) the necrotic (35.1 ± 1.2 vs. 29.7 ± 0.7) and viable sperm (37.2 ± 1.3 vs. 32.8 ± 1.9, (P < 0.07). Likewise, the mean percent motility and plasma membrane integrity decreased (P < 0.05) (64 ± 2.1 vs. 49.4 ± 1.3) and (79.6 ± 0.5 vs. 38.7 ± 0.3) respectively, at post thaw compared to other stages. Coefficient of correlation, combined at all stages for each variable revealed that sperm apoptosis was inversely correlated with sperm motility and plasma membrane integrity. It is concluded that (a) the annexin V/PI assay can be used as a tool to determine the buffalo semen apoptosis and (b) freezing and thawing induces apoptosis in buffalo sperm.     

Involvement of polyamines, abscisic acid and anti-oxidative enzymes in adaptation of Blue Panicgrass (Panicum antidotale Retz.) to saline environments

A hydroponic experiment was conducted to assess the possible involvement of polyamines (PAs), abscisic acid (ABA) and anti-oxidative enzymes such as superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) in adaptation of six populations of Panicum antidotale Retz. to selection pressure (soil salinity) of a wide range of habitats. Plants of six populations were collected from six different habitats with ECe ranging from 3.39 to 19.23 dS m⁻¹ and pH from 7.65 to 5.86. Young tillers from 6-month-old plants were transplanted in plastic containers each containing 10 l of half strength Hoagland's nutrient solution alone or with 150 mol m⁻³ NaCl. After 42 days growth, contents of polyamines (Put, Spd and Spm) and ABA, and the activities of anti-oxidative enzymes (SOD, POD and CAT) of all populations generally increased under salt stress. The populations collected from highly saline habitats showed a greater accumulation of polyamines and ABA and the activities of anti-oxidative enzymes as compared to those from mild or non-saline habitats. Moreover, Spm/Spd and Put/(Spd + Spm) ratios generally increased under salt stress. However, the populations from highly saline environments had significantly higher Spm/Spd and Put/(Spd + Spm) ratios as compared to those from mild or non-saline environments. Similarly, the populations adapted to high salinity accumulated less Na⁺ and Cl⁻ in culm and leaves, and showed less decrease in leaf K⁺ and Ca²⁺ under salinity stress. Higher activities of anti-oxidative enzymes and accumulation of polyamines and ABA, and increased Spm/Spd and Put/(Spm + Spd) ratios were found to be highly correlated with the degree of adaptability of Panicum to saline environment.     

Response of native grasses and Cicer arietinum to soil polluted with mining wastes: Implications for the management of land adjacent to mine sites

Mine tailings are an environmental problem in Southern Spain because wind and water erosion of bare surfaces results in the dispersal of toxic metals over nearby urban or agricultural areas. Revegetation with tolerant native species may reduce this risk. We grew two grasses, Lygeum spartum and Piptatherum miliaceum, and the crop species Cicer arietinum (chickpea) under controlled conditions in pots containing a mine tailings mixed into non-polluted soil to give treatments of 0%, 25%, 50%, 75% and 100% mine tailings. We tested a neutral (pH 7.4) mine tailings which contained high concentrations of Cd, Cu, Pb and Zn. Water-extractable metal concentrations increased in proportion to the amount of tailings added. The biomass of the two grasses decreased in proportion to the rate of neutral mine-tailing addition, while the biomass of C. arietinum only decreased in relation to the control treatment. Neutron radiography revealed that root development of C. arietinum was perturbed in soil amended with the neutral tailings compared to those of the control treatment, despite a lack of toxicity symptoms in the shoots. In all treatments and for all metals, the plants accumulated higher concentrations in the roots than in shoots. The highest concentrations occurred in the roots of P. miliaceum (2500 mg kg^?1 Pb, 146 mg kg^?1 Cd, 185 mg kg^?1 Cu, 2700 mg kg^?1 Zn). C. arietinum seeds had normal concentrations of Zn (70–90 mg kg^?1) and Cu (6–9 mg kg^?1). However, the Cd concentration in this species was ～1 mg kg^?1 in the seeds and 14.5 mg kg^?1 in shoots. Consumption of these plant species by cattle and wild fauna may present a risk of toxic metals entering the food chain.     

Role of regulatory agencies

In this paper the authors discuss the role of regulation in assuring blood safety. After an overview of the subject by a leading expert, examples are provided of regulatory systems for blood transfusion services in several countries and regions. Additionally, the perspective of WHO is given on the essential role of national regulatory authorities in assuring the quality of national blood programmes.Collectively, the sections of this paper afford an opportunity for readers to make comparisons among different regulatory frameworks and to "benchmark" among the existing systems. Despite many differences in approach, a clear pattern emerges of worldwide efforts to strengthen blood regulatory systems.     

A Novel Glucosyltransferase Involved in O-Antigen Modification of Shigella flexneri Serotype 1c

The O antigen of serotype 1c differs from the unmodified O antigen of serotype Y by the addition of a disaccharide (two glucosyl groups) to the tetrasaccharide repeating unit. It was shown here that addition of the first glucosyl group is mediated by the previously characterized gtrI cluster, which is found within a cryptic prophage at the proA locus in the bacterial chromosome. Transposon mutagenesis was performed to disrupt the gene responsible for addition of the second glucosyl group, causing reversion to serotype 1a. Colony immunoblotting was used to identify the desired revertants, and subsequent sequencing, cloning, and functional expression successfully identified the gene encoding serotype 1c-specific O-antigen modification. This gene (designated gtrIC) was present as part of a three-gene cluster, similar to other S. flexneri glucosyltransferase genes. Relative to the other S. flexneri gtr clusters, the gtrIC cluster is more distantly related and appears to have arrived in S. flexneri from outside the species. Analysis of surrounding sequence suggests that the gtrIC cluster arrived via a novel bacteriophage that was subsequently rendered nonfunctional by a series of insertion events.Shigella flexneri is a pathovar of Escherichia coli that is the main causative agent of endemic bacillary dysentery (shigellosis). It is estimated that S. flexneri is responsible for approximately 100 million shigellosis cases annually, resulting in hundreds of thousands of deaths, predominantly in young children (11). Currently no vaccine is available, although there is evidence to suggest that serotype-specific immunity occurs following infection and that induction of immunity can be replicated with vaccines (9). Shigella serotype diversity arises due to differences in the chemical structure of the O-antigen repeating unit in the lipopolysaccharide, which is the main target of the adaptive host immune response following infection.Because immunity to S. flexneri can be conferred by the induction of antibodies directed against the O antigen, an understanding of the prevalence of different serotypes and the underlying basis of serotype diversity can inform appropriate vaccine design. All S. flexneri serotypes (with the exception of serotype 6) share a common O-antigen backbone, consisting of a repeating tetrasaccharide unit that is comprised of one N-acetylglucosamine residue (GlcNAc) and three rhamnose residues (RhaI, RhaII, and RhaIII) (14). The 12 traditionally recognized S. flexneri serotypes differ by the presence or absence of just six different chemical modifications (glucosylations or O acetylations) of the O antigen. The genes responsible for these O-antigen modifications are introduced into the bacterial genome via bacteriophages (3). Glucosylation of the S. flexneri O antigen is mediated by three genes [gtrA, gtrB, and gtr(type)] that are arranged in a single operon known as a gtr cluster. gtrA and gtrB are highly conserved between different gtr clusters and encode proteins involved in transferring the glucosyl group from the cytoplasm into the periplasm, where O-antigen modification is thought to take place. gtr(type) is unique to each gtr cluster and encodes a glucosyltransferase that is responsible for attaching the glucosyl group to a specific sugar unit of the O antigen via a specific linkage (3).Investigations of S. flexneri have typically focused on serotypes for which commercially available typing sera are available. More recently, it has become clear that other serotypes are also epidemiologically important. In Bangladesh in the late 1980s, two novel S. flexneri strains that did not agglutinate with antibodies specific for the traditionally recognized serotypes were isolated (4). Chemical analysis of the O antigen revealed that these strains belonged to a new serotype, which was named serotype 1c due to the similarity its O antigen shares with the O antigens of serotype 1a and 1b strains (19). Serotype 1c has since been isolated in Egypt, Indonesia, Pakistan, and Vietnam (6, 15, 18). Serotype 1c was shown to be the most prevalent S. flexneri serotype in a northern province of Vietnam, accounting for more than a third of all S. flexneri strains isolated from 1998 to 1999 (15). Identification of serotype 1c currently relies on agglutination testing using monoclonal antibody MASF Ic (19).The O antigen of serotype 1c is distinguished by the presence of a disaccharide (two glucosyl groups) linked to the GlcNAc in the tetrasaccharide repeating unit of the O antigen. The first glucosyl group is joined to GlcNAc via an α1→4 linkage, as occurs in the O antigen of serotype 1a and serotype 1b strains (type I modification). The O antigen of serotype 1c is distinguished by the presence of a second glucosyl group that is linked to the first via an α1→2 linkage (Fig. ​(Fig.1).1). Type Ia modification is prerequisite to type Ic modification.Open in a separate windowFIG. 1.Chemical structure of the tetrasaccharide repeat units in the O antigens of S. flexneri serotypes 1a and 1c. Note that the O antigen of serotype 1b (not shown) differs from that of serotype 1a by the O acetylation of l-RhaIII.In this study, the genetic basis of O-antigen modification in serotype 1c was elucidated. Serotype 1c strains isolated from different locations and times were compared to gain insight into the evolution of this serotype. This is the first report of the identification of a glucosyltransferase gene that is responsible for addition of the second glucosyl group, causing serotype conversion from serotype 1a to serotype 1c.     

IGF-1 and G-CSF complement each other in BMSC migration towards infarcted myocardium in a novel in vitro model

Stem cell capability enhanced with cytokine administration is a promising treatment for myocardial infarction. Bone marrow stem cells (BMSCs) were isolated from C57BL/6 mice (8-12 weeks old) expressing GFP and characterized with c-kit and CD34. Infarcted heart tissue fragments were placed into dishes with BMSCs and medium supplemented with G-CSF, SCF, IGF-1 or combinations thereof were given to the BMSC-infarcted myocardium in vitro model. The IGF-1-G-CSF group showed significantly higher migration (67.7% ± 2.6) of c-kit⁺ BMSCs towards the ischemic tissue and expressed MEF-2 (43.7% ± 1.7). Of the single treatment groups, the G-CSF group demonstrated significantly higher migration of c-kit⁺ BMSCs (60.5 ± 2.7) with MEF-2 expression (38.7 ± 1.4). IGF-1 complements G-CSF and was relatively more significant in its effects on BMSC migration and cardiac lineage commitment towards ischemic heart tissue.