Generation of New Hairless Alleles by Genomic Engineering at the Hairless Locus in Drosophila melanogaster

Hairless (H) is the major antagonist within the Notch signalling pathway of Drosophila melanogaster. By binding to Suppressor of Hairless [Su(H)] and two co-repressors, H induces silencing of Notch target genes in the absence of Notch signals. We have applied genomic engineering to create several new H alleles. To this end the endogenous H locus was replaced with an attP site by homologous recombination, serving as a landing platform for subsequent site directed integration of different H constructs. This way we generated a complete H knock out allele H ^attP, reintroduced a wild type H genomic and a cDNA-construct (H ^gwt, H ^cwt) as well as two constructs encoding H proteins defective of Su(H) binding (H ^LD, H ^iD). Phenotypes regarding viability, bristle and wing development were recorded, and the expression of Notch target genes wingless and cut was analysed in mutant wing discs or in mutant cell clones. Moreover, genetic interactions with Notch (N ⁵⁴¹⁹) and Delta (Dl ^B2) mutants were addressed. Overall, phenotypes were largely as expected: both H ^LD and H ^iD were similar to the H ^attP null allele, indicating that most of H activity requires the binding of Su(H). Both rescue constructs H ^gwt and H ^cwt were homozygous viable without phenotype. Unexpectedly, the hemizygous condition uncovered that they were not identical to the wild type allele: notably H ^cwt showed a markedly reduced activity, suggesting the presence of as yet unidentified regulatory or stabilizing elements in untranslated regions of the H gene. Interestingly, H ^gwt homozygous cells expressed higher levels of H protein, perhaps unravelling gene-by-environment interactions.     

Biosynthesis of bacterial glycogen: purification and characterization of ADPglucose pyrophosphorylase with modified regulatory properties from Escherichia coli B mutant CL1136-504

The l-thyroxine binding site in human serum thyroxine-binding globulin was investigated by affinity labeling with N-bromoacetyl-l-thyroxine (BrAcT₄). Competitive binding studies showed that, in the presence of 100 molar excess of BrAcT₄, binding of thyroxine to thyroxine-binding globulin was nearly totally abolished. The reaction of BrAcT₄ to form covalent binding was inhibited in the presence of thyroxine and the affinity-labeled thyroxinebinding globulin lost its ability to bind thyroxine. These results indicate BrAcT₄ and thyroxine competed for the same binding site. Affinity labeling with 2 mol of BrAcT₄/mol of thyroxine-binding globulin resulted in the covalent attachment of 0.7 mol of ligand. By amino acid analysis and high voltage paper electrophoresis, methionine was identified as the major residue labeled (75%). Lysine, tyrosine, and histidine were also found to be labeled to the extent of 8, 8, and 5%, respectively.     

Normalized cDNA libraries from a porcine model of orthopedic implant-associated infection

Staphylococcal infections that result from an alteration in a patient's immune response at the surgical site are a major problem in procedures that incorporate biomaterials in trauma surgery and joint replacement. Diagnosis of infection based on pathogen detection is difficult and exacerbated by increasing numbers of partially or totally resistant strains of nosocomial pathogens, particularly Staphylococcus aureus. Expression profiling of a host's cellular immune response could facilitate the identification of the pathways involved in pathogen recognition and eradication and could lead to more rational design of drugs and therapies. To this end, we constructed and characterized ten individually tagged and directionally cloned cDNA libraries from peripheral blood cells (PBC), spleen (Sp), thymus (Th), lymph node (LN), and bone marrow (BM) from immunologically naive and challenged pigs as part of an implant-associated orthopedic model of deep infection. Three of these libraries were normalized at C _{0 t} values 5, 10, 20, and 30. The libraries comprise more than 20 million primary transformants with an average insert length >1.4 kb. Cluster analysis of 7620 ESTs revealed 1029 clusters containing an average of 3.6 sequences and 3846 singletons. Gene discovery is estimated to be ∼64%. Searches of public databases resulted in 49.3% annotated porcine sequences, of which 22.2% had significant homologies to ESTs from a variety of species, and 28.5% were without a significant match in any public database. We also identified 9.1% ESTs as involved in host cell and organism defense and 11.5% related to cell signaling and communication. These sequences, together with the 28.5% appearing as novel, are of specific interest to the infectious disease process.     

Cyclophosphamide and related anticancer drugs

This article presents an overview of the methods of bioanalysis of oxazaphosphorines, in particular, cyclophosphamide, ifosfamide, and trofosfamide as well as their metabolites. The metabolism of oxazaphosphorines is complex and leads to a large variety of metabolites and therefore the spectrum of methods used is relatively broad. The various methods used are shown in a table and the particularly important assays are described.     

Liquid chromatographic determination of the enantiomers of ibuprofen in plasma using a chiral AGP column

A reversed-phase high-performance liquid chromatographic method has been developed for the determination of the R- and S-enantiomers of ibuprofen. The enantiomers and the internal standard 4-pentylphenylacetic acid are extracted from plasma, separated and quantified on a Chiral-AGP column using ultraviolet detection. The simplicity, sensitivity and precision of the method makes it convenient for use in pharmacokinetic studies.     

Effect of external sodium and calcium on calcium efflux in arteria carotis of cattle

The dependence of the 45Ca-efflux from the smooth muscle cells of the arteria carotis of cattle on external sodium and calcium was studied. In the absence of external calcium the replacement of NaCl by sucrose leads to a decreased 45Ca-efflux rate, replacement by LiCl to an increased efflux rate. With regard to the presence of sodium and calcium in the external medium, the 45Ca-efflux rate decreases in the following order: Na + Ca less than less than Na + Ca-free less than Na-free (Na substituted by sucrose) + Ca-free. LiCl considerably stimulates the 45Ca-efflux rate in the presence of external calcium. An inhibition of the Na, K-ATPase activity lasting for more than 20 min leads to a decrease of the 45Ca-efflux rate. The results obtained suggest the existence of a Na-Ca-exchange in the arteria carotis of cattle.