Penicillin amidase-catalysed transfer of low specific acyl moiety. Synthesis of 7-benzoxazolonylacetamido desacetoxycephalosporanic acid.

The methyl ester of 2-benzoxazolon-3-yl-acetic acid was used as an acyl donor in the penicillin amidase-catalysed transfer reaction to 7-aminodesacetoxycephalosporanic acid. The synthesis of 7-(2-benzoxazolon-3-yl-acetamido)-desacetoxycephalosporanic acid was carried out as a kinetically controlled reaction. A characteristic feature of this system is that the benzoxazolone derivatives are very low specific substrates for penicillin amidase (the kcat/Km values for their hydrolysis were shown to be 10(5)-fold lower compared to the corresponding values for phenylacetyl derivatives). Nevertheless, penicillin amidase proved to be an effective catalyst for the synthesis of these new cephem derivatives (50% yield for 6 h). The reason is the observed unusually high value for the transferase-hydrolase activity ratio. The determined value for (k3'/k3)app = 120,000 implies that in this case of low specific acyl moiety, penicillin amidase acts more like a transferase than a hydrolase. The maximum yield has been increased up to 70% by lowering the reaction temperature and stepwise feeding of the reaction medium with the acyl component. The results obtained extend the potential of the penicillin amidase as a catalyst for the synthesis of a new group of biologically active cephem derivatives.     

The methylerythritol phosphate pathway for isoprenoid biosynthesis in coccidia: presence and sensitivity to fosmidomycin

The apicoplast is a recently discovered, plastid-like organelle present in most apicomplexa. The methylerythritol phosphate (MEP) pathway involved in isoprenoid biosynthesis is one of the metabolic pathways associated with the apicoplast, and is a new promising therapeutic target in Plasmodium falciparum. Here, we check the presence of isoprenoid genes in four coccidian parasites according to genome database searches. Cryptosporidium parvum and C. hominis, which have no plastid genome, lack the MEP pathway. In contrast, gene expression studies suggest that this metabolic pathway is present in several development stages of Eimeria tenella and in tachyzoites of Toxoplasma gondii. We studied the potential of fosmidomycin, an antimalarial drug blocking the MEP pathway, to inhibit E. tenella and T. gondii growth in vitro. The drug was poorly effective even at high concentrations. Thus, both fosmidomycin sensitivity and isoprenoid metabolism differs substantially between apicomplexan species.     

Immune regulation of protection and pathogenesis in Plasmodium falciparum malaria

The immune mechanisms whereby malaria parasites are eliminated by the human host or how they may avoid the immune response are poorly understood. Individuals living in malaria-endemic areas gradually acquire immunity. It is well established that this immunity involves both cell-mediated and humoral mechanisms and that T cells are the major regulators in both these events. The existence of functionally distinct P. falciparum-specific CD4+ T-cell subsets in humans has been shown in several studies. However, in contrast to what is the case in murine models there is no definitive link between the activation of various T cells and the course of human P. falciparum blood-stage infection. In the present paper we will review recent findings which illustrate how the balance between functionally different T-cell subsets affects the development of malaria immunity but also may contribute to its pathogenicity. An example of the latter is the deposition of IgE-containing immune complexes in small vessels, probably leading to local overproduction of tumor-necrosis factor (TNF), a pathogenic factor in malaria.     

Repetin (Rptn), a New Member of the “Fused Gene” Subgroup within the S100 Gene Family Encoding a Murine Epidermal Differentiation Protein

We report the cloning and characterization of a murine epidermal differentiation gene, repetin (Rptn), exhibiting striking similarity to the genes of the intermediate filament-associated proteins profilaggrin and trichohyalin. The repetin gene consists of three exons and two introns. The first exon is short and untranslated. The deduced amino acid sequence distributed between exons II and III contains 1130 amino acids with a calculated molecular mass of 130 kDa and pIof 7.7. The amino terminus exhibits significant homology to the S100 proteins containing two calcium-binding motifs of the EF-hand type. The remainder coding sequence contains a central segment consisting of 49 tandem repeats of a 12-amino-acid sequence rich in glutamines. By fluorescencein situhybridization the repetin gene was localized to chromosome band 3 F1-2. Expression of repetin mRNA is detectable in the stratified internal epithelia of forestomach and tongue and to a lesser degree in normal skin epidermis, where it is restricted to the differentiated suprabasal cell layers. Based on its chromosomal localization, its genomic organization, and its stage-specific expression during late epidermal differentiation, as well as on the structural features of the encoded protein, we conclude that the repetin gene represents a novel member of the “fused gene” subgroup of the S100 gene family encoding multifunctional epidermal matrix proteins.     

Synthesis, radiosynthesis and in vivo preliminary evaluation of [11C]LBT-999, a selective radioligand for the visualisation of the dopamine transporter with PET

LBT-999 (8-((E)-4-fluoro-but-2-enyl)-3beta-p-tolyl-8-aza-bicyclo[3.2.1]octane-2beta-carboxylic acid methyl ester), a cocaine derivative belonging to a new generation of highly selective dopamine transporter (DAT) ligands, and its corresponding carboxylic acid derivative, the latter used as precursor for labelling both with tritium and the positron-emitter carbon-11 (half-life: 20.38 min), were synthesized from (R)-cocaine. [(3)H]LBT-999 (>99% radiochemically pure, specific radioactivity of 3.1 TBq/mmol) was prepared from [(3)H]methyl iodide, allowing its in vitro pharmacological evaluation (K(D): 9 nM for DAT and IC(50) > 1000 nM for SERT and NET). Routine production batches of 4.5-9.0 GBq of iv injectable solutions of [(11)C]LBT-999 (with specific radioactivities ranging from 30 to 45 GBq/mumol) were prepared in 25-30 min (HPLC purification and formulation included) using the efficient methylation reagent [(11)C]methyl triflate. The preliminary in vivo pharmacological evaluation of [(11)C]LBT-999, using both biodistributions in rats and brain imaging in monkeys with positron emission tomography (PET), clearly illustrates that this ligand is an excellent candidate for quantification with PET of DAT in humans.