Novel stabilization of phenylalanine ammonia-lyase catalyst during bioconversion of trans-cinnamic acid to l-phenylalanine

Summary Production of l-phenylalanine from trans-cinnamic acid using isolate SPA10 cells was reduced to 26% of that observed initially when cells were reacted a second time with fresh substrate mixture. The stability (reuseability) of Phenylalanine Ammonia-Lyase (PAL) containing cells was significantly influenced by both the trans-cinnamate concentration and initial reaction pH. Using 2% t-cinnamate, l-phenylalanine production was 7-fold greater after 3 successive runs at pH 9.0 than at the optimum of pH 10.2. Cells reacted in the presence of 5% t-cinnamate were relatively unstable. Permeabilising agents, such as toluene and xylene, stimulated l-phenylalanine production but also enhanced instability of the catalyst. Several effectors were shown to stimulate the initial rate of the PAL bioconversion, but only sorbitol, alginate, glutaraldehyde, polyethylene glycol and glycerol conferred any significant degree of stability. Sparging of cultures and bioreactors with various gases revealed that oxygen enhanced PAL inactivation, CO₂ had little effect and nitrogen conferred remarkable stability on PAL activity for several weeks in culture medium. The presence of chloride ions (from HCl) and aeration of substrate mixtures resulted in poor reuseability of catalyst. A combination of H₂SO₄ substitution for HCl and N₂-sparging resulted in excellent initial conversions and good catalyst stability at 26°C but less at 30°C. The inclusion of 1.5 M sorbitol in reaction mixtures maintained PAL stability over several successive incubations.     

The codon 408 mutation associated with haplotype 2 is predominant in Polish families with phenylketonuria

Summary The incidence of phenylketonuria (PKU) in the western part of Poland is 1 in 5000 live births. Restriction fragment length polymorphism (RFLP) haplotypes at the phenylalanine hydroxylase locus have been analysed in 46 Polish families with PKU. Among 43 fully-informative families 16 RFLP haplotypes were identified. Haplotype 2 is the most frequently (62%) associated with Polish PKU alleles, and the codon 408 mutation is in complete linkage disequilibrium with this haplotype in Poland. This finding is in agreement with observations in other eastern European countries (German Democratic Republic, Czechoslovakia, and Hungary) and in contrast to the genotype distribution observed in western European countries. The present observation suggests the spread of classical PKU, due to the codon 408 mutation associated with haplotype 2, from east to west in European populations. Perhaps more important for genetic counselling, 62% of all PKU chromosomes in the Polish population can now be detected using only one mutantspecific oligonucleotide probe.     

The coupling of crossing over and homologous synapsis in maize inversions

Because fresh initiations of synapsis must occur for homologous synapsis of internal heterozygously inverted chromosome segments, attention has been directed at homologous synapsis and crossing over in overlapping paracentric inversions in the long arm of chromosome 1 of maize. In an earlier study with a relatively short inversion (where double crossovers within the inversion were rare), a recombination nodule (RN) was generally found at pachytene in reverse paired (homologously synapsed) inverted regions. Crossover frequency within the inversion, which could be independently estimated from analysis of bridge and fragment frequency at anaphase I and II, closely corresponded to crossover frequency estimated from observed RN frequency in pachytene inversion loops. These findings were consistent with the interpretation that establishment of homologous synapsis in this case is generally coupled to crossing over. This coupling suggests that there is very early commitment to the form of resolution of recombination intermediates that results in reciprocal recombination events instead of conversion only or other noncrossover events. This study examines another, larger paracentric inversion in the long arm of chromosome 1 that completely overlaps the first inversion. It is sufficiently longer than the first inversion that double crossover events are found within it with substantial frequency and interference considerations are feasible. This study confers additional insight into the interrelationships of synapsis and crossing over and the probable sequence in which the various involved processes usually occur. It raises the strong possibility that crossovers can be initiated during the alignment phase that precedes synapsis.     

Synaptic defects of asynaptic homozygotes in maize at the electron microscope level.

More detailed observations of the synaptonemal complex (SC) in asynaptic maize plants have been faciliated by superior silver-staining procedures. These suggest that central region components of the SC are strongly implicated as defective in asynaptic. Apparently homologous axial elements tend to follow roughly parallel courses within the nucleus at pachytene, in some short segments apparently synapsed and in others at wider separation than normal synapsis yet close enough to allow observation of thin central element segments and also occasional thin transverse element-type structures. This kind of transverse filament may be weakened and severely stretched yet associated with both axial elements. Small nodules, similar to recombination nodules, appear at corresponding positions in widely separated axial elements. Key words : synaptonemal complex, central element, transverse filament, recombination nodule.     

Chromosomal assignment of the human smg GDP dissociation stimulator gene to human chromosome 4q21-q25

The 3-end of the cDNA encoding the smg GDP dissociation stimulator (smg GDS) protein shares 100% homology with the previously published expressed sequence tag 00038 site. This site extends the 3-end of the smg GDS gene by 212 bp. It has been localized to human chromosome 4. Here, we have refined the localization of smg GDP to human chromosome 4q21-q25 using a mapping panel of rodent/human somatic cell hybrids containing different parts of chromosome 4. This chromosomal localization of smg GDP to 4q21-25 overlaps with a region of allele loss in primary hepatocellular carcinoma (4q13-q26).HGM symbol: RAP1GDS1     

Catalase anabolism in yeast: Loss of regulation by oxygen of catalase apoprotein synthesis after mutation

Summary A mutant of Saccharomyces cerevisiae which displays catalase activity when grown under strictly anaerobic conditions has been selected on solid media.Although some preformed holoenzyme has accumulated in anaerobic cells, a sharp increase of activity is still measured during adaptation to oxygen in glucose-buffer; however, a striking difference with the wild-type strain is that in the mutant, catalase formation is observed in the presence of cycloheximide that totally inhibits cytoplasmic translation. It is concluded that kat 80 mutant has lost the regulatory control by oxygen of apocatalase synthesis; the latter precursor, characterized as apocatalase T, is thought to be activated in vivo, under aerobic conditions, by inclusion of prosthetic group.Regulation of enzyme synthesis by catabolite repression (glucose effect) persists, unmodified by reference to the wild-type parental strain.Mutation kat 80 specifically hits catalase anabolism, as no significant variations were observed for the edification of the respiratory system and (apo)cytochrome c peroxidase production.Genetic analysis shows that kat 80 phenotype, recessive in heterozygotes, results from a single nuclear mutation.Abbreviations Enzymes. Catalase or hydrogen-peroxide hydrogen-peroxide oxidoreductase (EC 1.11.1.6) - Cytochrome c peroxidase or ferrocytochrome c hydrogen-peroxide oxidoreductase (EC 1.11.1.5)     

Origin of Haemophilus influenzae R factors.

The Haemophilus influenzae R plasmids specifying resistance against one, two, or three antibiotics which have emerged in different parts of the world were shown to have closely related but not identical plasmid cores. The gene for ampicillin resistance in the H. influenzae plasmid pKRE5367 is part of a transposon similar to Tn3, which was transposed from pKRE5367 onto RSF1010 in Escherichia coli. An indigenous H. influenzae plasmid (pW266) was isolated. Its properties correspond to those of the H. influenzae R plasmids, except for the presence of a drug resistance transposon. The in vitro-generated H. influenzae R plasmids carrying an ampicillin resistance transposon, a tetracycline resistance transposon, and a transposon for combined tetracycline-chloramphenicol resistance resembled the natural isolates. The findings support the hypothesis that the R plasmids of H. influenzae are of multiclonal evolutionary origin.     

Improvement of pneumonia and arthritis in Felty's syndrome by treatment with granulocyte-macrophage colony-stimulating factor (GM-CSF)

A 63-year old man with Felty's syndrome and pneumonia of unknown origin was treated with GM-CSF. Granulocyte counts increased and arthritis-related symptoms improved under GM-CSF. Pneumonia was treated effectively with antibiotics only during or after GM-CSF application. This suggests, that antibiotic-resistant infections can be treated effectively in patients with Felty's syndrome when granulocyte counts are raised by GM-CSF.