Determination of full 13C isotopomer distributions for metabolic flux analysis using heteronuclear spin echo difference NMR spectroscopy.

13C-isotopomer labeling experiments play an increasingly important role in the analysis of intracellular metabolic fluxes for genetic engineering purposes. 13C NMR spectroscopy is a key technique in the experimental determination of isotopomer distributions. However, only subsets of isotopomers can be quantitated using this technique due to redundancies in the scalar coupling patterns and due to invisibility of the 12C isotope in NMR. Therefore, we developed and describe in this paper a 1H NMR spectroscopy method that allows to determine the complete isotopomer distribution in metabolites having a backbone consisting of up to at least four carbons. The proposed pulse sequences employ up to three alternately applied frequency-selective inversion pulses in the 13C channel. In a first application study, the complete isotopomer distribution of aspartate isolated from [1-13C]ethanol-grown Ashbya gossypii was determined. A tentative model of the central metabolism of this organism was constructed and used for metabolic flux analysis. The aspartate isotopomer NMR data played a key role in the successful determination of the flux through the peroxisomal glyoxylate pathway. The new NMR method can be highly instrumental in generating the data upon which isotopomer labeling experiments for flux analysis, that are becoming increasingly important, are based.

[Initiation of treatment following screening for phenylketonuria]. / Behandlungsbeginn nach der Vorsorgeuntersuchung auf Phenylketonurie.

The newborn screening for PKU is widely established in the F.R.G since 1969. Apart from the quality of dietary control, the age at starting therapy seems to be of high importance for the normal development of these patients. Therefore, the steps from first recording of an elevated Phe level until the beginning of treatment are listed and both, the optimal and the usual time procedure --that is without errors or mishaps--are described. The data of the PKU-Collaborative Study serve to exemplify these courses of events. Reasons for delay are discussed und suggestions for its avoidance are made.

Cryptosporidium parvum is cytopathic for cultured human biliary epithelia via an apoptotic mechanism.

While the clinical features of sclerosing cholangitis secondary to opportunistic infections of the biliary tree in patients with acquired immunodeficiency syndrome (AIDS) are well known, the mechanisms by which microbial pathogens such as Cryptosporidium parvum associated with this syndrome actually cause disease are obscure. We established an in vitro model of biliary cryptosporidiosis employing a human biliary epithelial cell line. Using morphological and biochemical techniques, we examined the interaction of C. parvum with cultured human cholangiocytes. When the apical plasma membrane of polarized, confluent monolayers of human biliary epithelial cells was exposed to C. parvum oocysts that had been excysted in vitro, sporozoites attached to and invaded the cells in a time-, dose-, temperature-, and pH-dependent manner. The infectious process was both plasma membrane domain- and cell-specific, because no attachment or invasion occurred when the basolateral membrane of cholangiocytes was exposed to the parasite, or when a human hepatocyte cell line (HepG2) was used. Time-lapse video microscopy and scanning electron microscopy (SEM) showed that sporozoite attachment was rapid, involved extensive cholangiocyte membrane ruffling, and culminated in parasite penetration into a tight-fitting vacuole formed by invagination of the plasma membrane similar to those found in naturally occurring infection in vivo. Transmission electron microscopy (TEM) showed that C. parvum organisms formed parasitophorus vacuoles and were able to undergo a complete reproductive cycle, forming both asexual and sexual reproductive stages. Unexpectedly, direct cytopathic effects were noted in infected monolayers, with widespread programmed cell death (i.e., apoptosis) of biliary epithelial cells as assessed both morphologically and biochemically beginning within hours after exposure to the organism. The novel finding of specific cytopathic invasion of biliary epithelia by C. parvum may be relevant to the pathogenesis and possible therapy of the secondary sclerosing cholangitis seen in AIDS patients with biliary cryptosporidiosis.