Continuous, real-time monitoring of the oxygen uptake rate (OUR) in animal cell bioreactors

A new method for real-time monitoring of the oxygen uptake rate (OUR) in bioreactors, based on dissolved oxygen (DO) measurement at two points, has been developed and tested extensively. The method has several distinct advantages over known techniques.It enables the continuous and undisturbed monitoring of OUR, which is conventionally impossible without gas analyzers. The technique does not require knowledge of k(L)a. It provides smooth, robust, and reliable signal. The monitoring scheme is applicable to both microbial and mammalian cell bioprocesses of laboratory or industrial scale. The method was successfully used in the cultivation of NSO-derived murine myeloma cell line producing monoclonal antibody. It was found that while the OUR increased with the cell density, the specific OUR decreased to approximately one-half at cell concentrations of 16 x 10(6) cells/mL, indicating gradual reduction of cell respiration activity. Apart from the laboratory scale cultivation, the method was applied to industrial scale perfusion culture, as well as to processes using other cell lines. (c) 1994 John Wiley & Sons, Inc.     

Identification of RFLP markers closely linked to the bolting gene B and their significance for the study of the annual habit in beets (Beta vulgaris L.)

The annual habit in beet is due to complete or partial absence of the vernalization requirement and can cause severe problems in the beet crop. The absolute vernalization requirement in beet is controlled by a major geneB (bolting), known to be linked to the geneR (red hypocotyl color), in linkage group I. Segregation for theB andR genes was studied in several beet progenies. Penetrance of the annual habit inBb genotypes was affected by both environmental and genetic factors. The precise location in linkage group I of the major geneB was found by restriction fragment length polymorphism (RFLP) analysis in a back-cross progeny exhibiting partial penetrance of the annual habit. The linkage value betweenB andR was in good accordance with previous estimations. Use of the closest RFLP marker (pKP591: 3.8 recombination units) allowed us to estimate the penetrance of the annual habit in this back-cross as 0.62. Evidence of pseudo-compatibility was found in the wild coastal beet (Beta vulgaris sspmaritima) used as the mother plant of the back-cross: the selfing rate was estimated as 7%.     

Pool sequencing of natural HLA-DR,DQ, and DP ligands reveals detailed peptide motifs,constraints of processing,and general rules

We have approached the problem of MHC class II ligand motifs by pool sequencing natural peptides eluted from HLA-DR, DQ, and DP molecules. The results indicate surprisingly clear patterns, although not quite as clear as with natural class I ligands. The most striking feature is a highly dominant Proline at position 2. We interpret this to be a consequence of aminopeptidase N-like activity in processing. Another general aspect is the existence of three to four hydrophobic or aromatic anchors, whereby the first and the last are separated by five to eight residues. The peptide motifs for HLA-DR1, DR5, DQ7, and DPw4 are allele-specific and differ by spacing and occupancy of anchors. The anchors tend to be flanked by clusters of charged residues, and small residues, especially Ala, are frequent in the motif centers. These detailed motifs allow one to interpret most previous (DR-) motifs as fitting one or more of the anchors or conserved clusters. The relative motif symmetry suggests the possibility of bidirectional binding of peptides in the class II groove.     

Cation transport systems in mitochondria: Na+ and K+ uniports and exchangers

It is now well established that mitochondria contain three antiporters that transport monovalent cations. A latent, allosterically regulated K⁺/H⁺ antiport appears to serve as a cation-extruding device that helps maintain mitochondrial volume homeostasis. An apparently unregulated Na⁺/H⁺ antiport keeps matrix [Na⁺] low and the Na⁺-gradient equal to the H⁺-gradient. A Na⁺/Ca²⁺ antiport provides a Ca²⁺-extruding mechanism that permits the mitochondrion to regulate matrix [Ca²⁺] by balancing Ca²⁺ efflux against influx on the Ca²⁺-uniport. All three antiports have well-defined physiological roles and their molecular properties and regulatory features are now being determined. Mitochondria also contain monovalent cation uniports, such as the recently described ATP- and glibenclamide-sensitive K⁺ channel and ruthenium red-sensitive uniports for Na⁺ and K⁺. A physiological role of such uniports has not been established and their properties are just beginning to be defined.     

Rapid screening of an SH2-containing gene using PCR amplification method

Summary To isolate a novel gene that contains an SH2 domain, we devised a rapid and nonradioactive cDNA library screening method using polymerase chain reaction (PCR). For PCR amplification, we designed degenerate oligonucleotide primers from the multialigned DNA sequences of SH2 domains. This method offers an inexpensive and efficient approach for the isolation of clones of interest from cDNA libraries.     

Acceptability of carrier screening for cystic fibrosis during pregnancy in a German population

A pilot project offering voluntary heterozygote screening for the F508 mutation causing cystic fibrosis (CF) to 638 pregnant women attending two antenatal clinics in the eastern part of Berlin was carried out from 1990–1993. Participation was invited using an information leaflet and inclusion in the study was conditional on written informed consent. Of those invited to participate, only one refused to be tested, on the grounds of non-acceptance of prenatal diagnosis. Eighteen pregnant women were identified as carriers of the F508 mutation. All of them and their male partners accepted counselling in which the genetics of CF, its prognosis and treatment were explained, with emphasis on the meaning of heterozygosity, the fact that carriers are healthy, and the risk of an affected fetus when only one parent is identified as a heterozygote. All partners agreed to be tested for the F508 R553X and G551D mutations and a second counselling session was carried out after this test result was available. No problems were observed during initial testing but, as in other studies, we found considerable anxiety on being given the result in all couples where the woman tested positive; this was reduced substantially by counselling and when the partner tested negative. All probands found to be carriers stated that they found screening acceptable. In contrast to the cautious statement by the German Berufsverband Medizinische Genetik and the hostile reaction from a representative of the CF self-support organisation towards community-based heterozygote screening for CF, this study shows that CF screening is generally acceptable in this German population and that it is actively taken up by most pregnant women when offered.     

Localization of a New Type of X-Linked Liver Glycogenosis to the Chromosomal Region Xp22 Containing the Liver α-Subunit of Phosphorylase Kinase (PHKA2)

We describe here a new type of X-linked liver glycogen storage disease. The main symptoms include liver enlargement and growth retardation. The clinical and biochemical abnormalities of this glycogenosis are similar to those of classical X-linked liver glycogenosis due to phosphorylase kinase deficiency (XLG). However, in contrast to patients with XLG, the patients described here have no reduced phosphorylase kinase activity in erythrocytes and leukocytes, and no enzyme deficiency could be found. Linkage analysis of four families with this X-linked type of liver glycogenosis assigned the disease gene to Xp22. Lod scores obtained with the markers DXS987, DXS207, and DXS999 were 3.97, 2.71, and 2.40, respectively, all at 0% recombination. Multipoint linkage analysis localized the disease gene between DXS143 and DXS989 with a maximum lod score of 4.70 at θ = 0, relative to DXS987. As both the classical XLG gene and the liver α-subunit of PHK (PHKA2) are also located in Xp22, this variant type of XLG may be allelic to classical XLG, and both diseases may be caused by mutations in PHKA2. Therefore, we propose to classify XLG as XLG type I (the classical type of XLG) and XLG type II (the variant type of XLG).     

Enantiomerization of 2,2′-diisopropylbiphenyl during chiral inclusion gas chromatography: Determination of the rotational energy barrier by computer simulation of dynamic chromatographic elution profiles

By computer simulation of experimental dynamic gas chromatographic elution profiles, the rotational energy barrier ΔG⁼ of racemic 2,2′-diisopropylbiphenyl has been determined as 114.6–115.0 kJ/mol (75–100°C). These data are in good agreement with a value that was determined previously by measuring the racemization kinetics of an enriched sample. This indicates that there is no measurable catalytic or inhibitory effect of the stationary phase. © 1994 Wiley-Liss, Inc.