Myosin light-chain expression during avian muscle development

Monoclonal antibodies to adult chicken myosin light chains were generated and used to quantitate the types of myosin light-chain (MLC) isoforms expressed during development of the pectoralis major (PM), anterior latissimus dorsi (ALD), and medial adductor (MA) muscles of the chicken. These are muscles which, in the adult, are composed predominantly of fast, slow, and a mixture of fiber types, respectively. Three distinct phases of MLC expression characterized the development of the PM and MA muscles. The first identifiable pase occurred during the period of 5-7 d of incubation in ovo. Extracts of muscles from the pectoral region (which included the presumptive PM muscle) contained only fast MLC isoforms. This period of exclusive fast light-chain synthesis was followed by a phase (8- 12 d of incubation in ovo) in which coexpression of both fast and slow MLC isoforms was apparent in both PM and MA muscles. During the period, the composition of both fast and slow MLC isoforms in the PM and MA muscles was identical. Beginning at day 12 in ovo, the ALD was also subjected to immunochemical analyses. The proportion of fast and slow MLCs in this muscle at day 12 was similar to that present in the other muscles studied. The third development phase of MLC expression began at approximately 12 d of incubation in ovo and encompassed the transition in MLC composition to the isoform patterns incubation in ovo and encompassed the transition in MLC composition to the isoform patterns typical of adult muscle. During this period, the relative proportion of slow MLC rose in both the MA and ALD and fell in the PM. By day 16, the third fast light chain, LC(3f), was apparent in extracts of both the PM and MA. These results show that there is a developmental progression in the expression of MLC in the two avian muscles studied from day 5 in ovo; first, only fast MLCs are accumulated, then both fast and slow MLC isoforms are expressed. Only during the latter third of development in ovo is the final MLC isoform pattern characteristic of a particular muscle type expressed.     

In vitro effects of levamisole and ivermectin against Echinococcus granulosus protoscoleces

The in vitro effects of levamisole and ivermectin against Echinococcus granulosus protoscoleces were studied by means of light and electron microscopy. Both drugs had a protoscolicidal activity that increased proportionally with increasing concentrations of the drugs. Ivermectin showed the more rapid effects and caused contraction and paralysis of protoscoleces. A paralyzing effect was also observed with levamisole, followed by irreversible tissue vacuolation leading to death.     

Expression of surface hydrophobicity encoded by R-plasmids in Escherichia coli laboratory strains

The inclusion of drug-resistance plasmids (R-plasmids) in Escherichia coli strains has been shown to determine the formation of specific surface structures which could modify bacterial surface characteristics relevant for pathogenic processes.Thirtyone R-plasmids (from different incompatibility groups) have been transferred to three E. coli laboratory strains, and surface hydrophobicity modifications have been measured by three methods: salting-out, adsorption to hexadecane and adsorption to xylene.The results obtained show that the presence of R-plasmids produced variations which are dependent on the receptor strains and measuring method employed. Also, it has been found that the plasmids behave differently depending on the strain in which they are included.The results obtained by the salting-out method are not correlative with those obtained by adsorption to hydrocarbons, probably due to the implication of different hydrophobic molecules in the interaction with salt or hydrocarbons.Concluding, the choice of receptor strain and measuring method are of great importance for the investigation of surface hydrophobicity (and probably other characteristics) encoded by R-plasmids.Abbreviations TSB Trypticase soya broth - TSA trypticase soya agar     

Synthesis and characterization of Sodium cis-dichlorochenodeoxycholylglycinato(O,N) platinum(II) – Cytostatic activity

With a view to using bile acids as shuttles for delivering platinum-related cytostatic drugs to liver tumors, a chenodeoxycholylglycinato(CDCG)-derivative of platinum(II) has been synthesized. The complex - named Bamet-M2- was chemically characterized by elemental analysis, FT-IR, NMR, FAB-MS, and UV spectroscopy. The results indicate the following composition: C₂₆H₄₂N₂O₅Cl₂NaPt(II), the metal Pt(II) being bound to two Cl^– and one bidentate CDCG moiety, i.e., Na[Pt CDCG(N,O) Cl₂]. The compound is highly soluble (up to 20 mM) in water and (up to 35 mM) in ethanol, methanol and DMSO. Hydrolysis was investigated because this is assumed to be an important step in intracellular activation and interaction with DNA of this type of compounds. The reaction kinetics of this complex in aqueous solution show unusual behaviour; the substitution process with the displacement of two Cl^– was almost instantaneous, and the resulting species were found to be very stable. Kinetic studies carried out in the presence of different NaCl concentrations (up to 500 mM) revealed similar fast and nonreversible aquations of Bamet-M2. This contrasts with the slow aquation of cisplatin in extracellular-line solutions (i.e., at high NaCl concentrations) as compared with fast hydrolysis in cells. This difference may partly account for the low cytostatic activity observed here for Bamet-M2 against several tumor cell-lines.     

Visible-light promoted degradation of the commercial antioxidants butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT): a kinetic study

Visible-light photo-irradiation of the commercial phenolic antioxidants (PhAs) butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT), in the presence of vitamin B2 (riboflavin, Rf), in methanolic solutions and under aerobic conditions, results in the photo-oxidation of the PhAs. The synthetic dye photosensitiser Rose Bengal was also employed for auxiliary experiments. With concentrations of riboflavin and PhAs of ca. 0.02 mM and < 1 mM, respectively, the excited triplet state of the vitamin (3Rf*) is quenched by BHT in a competitive fashion with dissolved ground state triplet oxygen. From the quenching of 3Rf*, the semireduced form of the pigment is generated through an electron transfer process from BHT, with the subsequent production of superoxide anion radical (O2*-) by reaction with dissolved molecular oxygen. In parallel, the species singlet molecular oxygen, O2(1delta(g)), is also generated. Both reactive oxygen species produce the photodegradation of BHT. In the case of BHA, the lack of any effect exerted by superoxide dismutase drives out a significant participation of a O2(*-)-mediated mechanism. BHA mainly interacts with O2(1delta(g)) and exhibits a desirable property as an antioxidant--a relatively high capacity for O2(1delta(g)) de-activation and a low photodegradation efficiency by the oxidative species. Electrochemical determinations support the proposed photodegradative mechanism.     

Immunochemical Characterization of Temperature-Regulated Production of Enterocin L50 (EntL50A and EntL50B), Enterocin P, and Enterocin Q by Enterococcus faecium L50

Polyclonal antibodies with specificity for enterocin L50A (EntL50A), enterocin L50B (EntL50B), and enterocin Q (EntQ) produced by Enterococcus faecium L50 have been generated by immunization of rabbits with chemically synthesized peptides derived from the C terminus of EntL50A (LR1) and EntL50B (LR2) and from the complete enterocin Q (EntQ) conjugated to the carrier protein keyhole limpet hemocyanin (KLH). The sensitivity and specificity of these antibodies were evaluated by a noncompetitive indirect enzyme-linked immunosorbent assay (NCI-ELISA) and a competitive indirect ELISA (CI-ELISA). The NCI-ELISA was valuable for detecting anti-EntL50A-, anti-EntL50B-, and anti-EntQ-specific antibodies in the sera of the LR1-KLH-, LR2-KLH-, and EntQ-KLH-immunized animals, respectively. Moreover, these antibodies and those specific for enterocin P (EntP) obtained in a previous work (J. Gutiérrez, R. Criado, R. Citti, M. Martín, C. Herranz, M. F. Fernández, L. M. Cintas, and P. E. Hernández, J. Agric. Food Chem. 52:2247-2255, 2004) were used in an NCI-ELISA to detect and quantify the production of EntL50A, EntL50B, EntP, and EntQ by the multiple-bacteriocin producer E. faecium L50 grown at different temperatures (16 to 47°C). Our results show that temperature has a strong influence on bacteriocin production by this strain. EntL50A and EntL50B are synthesized at 16 to 32°C, but production becomes negligible when the growth temperature is above 37°C, whereas EntP and EntQ are synthesized at temperatures ranging from 16 to 47°C. Maximum EntL50A and EntL50B production was detected at 25°C, while EntP and EntQ are maximally produced at 37 and 47°C, respectively. The loss of plasmid pCIZ1 (50 kb) and/or pCIZ2 (7.4 kb), encoding EntL50A and EntL50B as well as EntQ, respectively, resulted in a significant increase in production and stability of the chromosomally encoded EntP.     

Using the ratio of means as the effect size measure in combining results of microarray experiments

Background  

Development of efficient analytic methodologies for combining microarray results is a major challenge in gene expression analysis. The widely used effect size models are thought to provide an efficient modeling framework for this purpose, where the measures of association for each study and each gene are combined, weighted by the standard errors. A significant disadvantage of this strategy is that the quality of different data sets may be highly variable, but this information is usually neglected during the integration. Moreover, it is widely known that the estimated standard deviations are probably unstable in the commonly used effect size measures (such as standardized mean difference) when sample sizes in each group are small.     

The loop between β-strands β2 and β3 and its interaction with the N-terminal α-helix is essential for biogenesis of α7 nicotinic receptors

Recently, we have shown that the α-helix present at the N-termini of α7 nicotinic acetylcholine receptors plays a crucial role in their biogenesis. Structural data suggest that this helix interacts with the loop linking β-strands β2 and β3 (loop 3). We studied the role of this loop as well as its interaction with the helix in membrane receptor expression. Residues from Asp62 to Val75 in loop 3 were mutated. Mutations of conserved amino acids, such as Asp62, Leu65 and Trp67 abolished membrane receptor expression in Xenopus oocytes. Others mutations, at residues Asn68, Ala69, Ser70, Tyr72, Gly74, and Val 75 were less harmful although still produced significant expression decreases. Steady state levels of wild-type and mutant α7 receptors (L65A, W67A, and Y72A) were similar but the formation of pentameric receptors was impaired in the latter (W67A). Mutation of critical residues in subunits of heteromeric nicotinic acetylcholine receptors (α3β4) also abolished their membrane expression. Complementarity between the helix and loop 3 was evidenced by studying the expression of chimeric α7 receptors in which these domains were substituted by homologous sequences from other subunits. We conclude that loop 3 and its docking to the α-helix is an important requirement for receptor assembly.     

Scavenging of photogenerated oxidative species by antimuscarinic drugs: atropine and derivatives

The quenching ability of photogenerated oxidative species by some antimuscarinic drugs generically named atropines (e.g. atropine [I] eucatropine [II], homatropine [III] and scopolamine [IV]) have been investigated employing stationary photolysis, polarographic detection of dissolved oxygen, stationary and time-resolved fluorescence spectroscopy, and laser flash photolysis. Using Rose Bengal as a dye sensitiser for singlet molecular oxygen, O(2)((1)Delta(g)), generation, compounds I-IV behave as moderate chemical plus physical quenchers of the oxidative species. Correlation between kinetic and electrochemical data indicates that the process is possibly driven by a charge-transfer interaction. The situation is somewhat more complicated employing the natural pigment riboflavin (Rf) as a sensitiser. Compounds I and II complex Rf ground state, diminishing the quenching ability towards singlet and triplet excited state of the pigment. On the other hand, compounds III and IV effectively quench Rf excited states, protecting the pigment against photodegradation. Under anaerobic conditions, semireduced Rf (Rf(.-)) is formed through quenching of excited triplet Rf. Nevertheless, although Rf(.-) is a well-known generator of the reactive species superoxide radical anion by reductive quenching in the presence of oxygen, the process of O(2)((1)Delta(g)) production prevails over superoxide radical generation, due to the relatively low rate constants for the quenching of triplet Rf by the atropines (in the order of 10(7) M(-1)s(-1) for compounds III and IV) in comparison to the rate constant for the quenching by ground state oxygen, approximately two orders of magnitude higher, yielding O(2)((1)Delta(g)). Compound I is the most promising O(2)((1)Delta(g)) physical scavenger, provided that it exhibits the higher value for the overall quenching rate constant and only 11% of the quenching process leads to its own chemical damage.