DETERMINATION OF GUSTATORY SUCROSE THRESHOLD IN WATER BY USE OF A LINEAR SUCROSE GRADIENT

A novel experimental method was developed which allows the determination of the threshold concentration of sucrose by use of a linear sucrose gradient in water. With this method a continuous tasting of the test-liquid is possible. A panel of 15 persons experienced in taste-testing was used. Three gradients of different steepness were applied: 0 to 1.5% (w/w) sucrose in 2 min (I), 3 min (II) and 4 min (III). The results of the new method were compared with those of the standard method (DIN). With gradients I and II we found values which were significantly higher than those of the standard method (I: 0.49% (w/w); II: 0.46% (w/w); DIN: 0.31% (w/w)), whereas with gradient III the same threshold value was found as with the DIN-Method (III: 0.32% (w/w)).     

Role of glycosylation on the secretion and biological activity of erythropoietin.

The erythropoietin (EPO) molecule contains four carbohydrate chains. Three contain N-linkages to asparagines at positions 24, 38, and 83, and one contains an O-linkage to a serine at position 126. We constructed human EPO variants that eliminated the three N-glycosylation sites by replacing the asparagines with glutamines singly or in combination. The O-linked carbohydrate chain was removed by replacing the serine with glutamine, valine, histidine, or alanine. A variant with a double mutation (Gln38,83) and another with a triple mutation (Gln24,38,83) were secreted poorly from COS1 and CHO cells even though RNA encoding these variants was present. All other variants with mutations in N-linked glycosylation sites were secreted normally. Removal of any of the N-glycosylation sites reduced the in vivo but not the in vitro biological activity of the EPO molecule. All the mutations at Ser126, the O-glycosylation site, were secreted normally. In vitro activity was also unaffected except for Ala126 which had a 50-fold decrease. The Val126 variant was tested in vivo, and its specific activity was only slightly less than that of the native EPO, which indicates that the O-linked carbohydrate is not essential for activity.     

Clues from a halophilic methanogen about aromatic amino acid biosynthesis in archaebacteria

Extensive diversity in features of aromatic amino acid biosynthesis and regulation has become recognized in eubacteria, but almost nothing is known about the extent to which such diversity exists within the archaebacteria. Methanohalophilus mahii, a methylotrophic halophilic methanogen, was found to synthesize l-phenylalanine and l-tyrosine via phenylpyruvate and 4-hydroxyphenylpyruvate, respectively. Enzymes capable of using l-arogenate as substrate were not found. Prephenate dehydrogenase was highly sensitive to feedback inhibition by l-tyrosine and could utilize either NADP⁺ (preferred) or NAD⁺ as cosubstrate. Tyrosine-pathway dehydrogenases having the combination of narrow specificity for a cyclohexadienyl substrate but broad specificity for pyridine nucleotide cofactor have not been described before. The chorismate mutase enzyme found is a member of a class which is insensitive to allosteric control. The most noteworthy character state was prephenate dehydratase which proved to be subject to multimetabolite control by feedback inhibitor (l-phenylalanine) and allosteric activators (l-tyrosine, l-tryptophan, l-leucine, l-methionine and l-isoleucine). This interlock type of prephenate dehydratase, also known to be broadly distributed among the gram-positive lineage of the eubacteria, was previously shown to exist in the extreme halophile, Halobacterium vallismortis. The results are consistent with the conclusion based upon 16S rRNA analyses that Methanomicrobiales and the extreme halophiles cluster together.Abbreviation DAHP 3-deoxy-d-arabino-heptulosonate-7-phosphate     

Sites of iodination in recombinant human brain-derived neurotrophic factor and its effect on neurotrophic activity.

Recombinant human brain-derived neurotrophic factor (BDNF) is now under extensive investigation because of its potential clinical applications. Radioactively labeled proteins are usually required to study receptor binding and pharmacokinetic properties of proteins. This study was undertaken to see if iodination affects the biological and conformational properties of a recombinant BDNF. BDNF was iodinated using a stoichiometric amount of nonradioactive cold NaI to minimize multiple iodinations. Of the four tyrosines present in BDNF--Tyr-52, Tyr-54, Tyr-63, and Tyr-86--only Tyr-63 and Tyr-86 were iodinated under the experimental conditions used. Iodination of Tyr-63 resulted in modification without alteration of the biological activity, whereas iodination of Tyr-86 resulted in a molecule with highly compromised biological activity. Similar inactivation was observed if both Tyr-63 and Tyr-86 were iodinated. These modified proteins exhibited conformation and dimerization apparently identical to those of the native protein, as demonstrated by analytical ultracentrifugation, gel filtration, light scattering, and circular dichroism. From these results, we concluded that Tyr-52 and Tyr-54 are not accessible to the reagent and are probably buried in the hydrophobic core, whereas Tyr-63 and Tyr-86 are exposed on the surface of the molecule; of the two exposed residues, only Tyr-86 contributes to the biological activity.     

Mixed-Culture Fermentor for Simulating Methanogenic Digestors

Propionate degradation in an anaerobic digestor degrading animal waste (10-day retention time, 5.75 g liter⁻¹ day⁻¹ volatile solids loading rate, 40°C) was 0.304 mM h⁻¹, measured with [2-¹⁴C]propionate; this value indicated that CH₄ produced from propionate accounted for 14.8% of the CH₄ produced in the digestor (34.5%, including acetate produced from propionate). The mean propionate concentration was 0.67 mM, giving a propionate turnover rate of 0.46 h⁻¹. A continuous-, mixed-culture fermentor was developed to mimic the digestor. When degradation rates of methanogenic precursors (H₂, CO₂, and acetate) equalled those measured in the digestor, propionate degradation was inhibited. When the H₂ turnover rate was lowered by decreasing addition of H₂-generating substrates or by allowing a portion of the H₂ degradation to occur in an isolated compartment, propionate degradation in the fermentor resumed. The possibility is discussed that in digestors, much of the H₂ is produced and degraded within microenvironments associated with particles. Thus, the gross turnover rate of H₂ measured in digestors is an average, and specific microenvironments within the digestor may have different rates of turnover.     

Yeast Kre2 Defines a New Gene Family Encoding Probable Secretory Proteins, and Is Required for the Correct N-Glycosylation of Proteins

We have cloned, sequenced and disrupted the KRE2 gene of Saccharomyces cerevisiae, identified by killer-resistant mutants with a defective cell wall receptor for the toxin. The KRE2 gene is close to PHO8 on chromosome 4, and encodes a predicted 49-kD protein, Kre2p, that probably enters the secretory pathway. Haploid cells carrying a disruption of the KRE2 locus grow more slowly than wild-type cells at 30 degrees, and fail to grow at 37 degrees. At 30 degrees, kre2 mutants showed altered N-linked glycosylation of proteins, as the average size of N-linked outer chains was reduced. We identified two other genes, YUR1 on chromosome 10, and KTR1 on chromosome 15, whose predicted products share 36% identity with Kre2p over more than 300 amino acid residues. Yur1p has an N-terminal signal sequence like Kre2p, while Ktr1p has a predicted topology consistent with a type 2 membrane protein. In all cases the conserved regions of these proteins appear to be on the lumenal side of secretory compartments, suggesting related function. KRE2, KTR1 and YUR1 define a new yeast gene family.     

Chemoprevention clinical trials.

As part of a program to develop drugs which will delay or prevent cancer in humans, the Chemoprevention Branch, National Cancer Institute, National Institutes of Health, is sponsoring 12 Phase I and 22 Phase II and III clinical trials. Three agent classes are significantly advanced in the trials. These are the retinoids, including 13-cis-retinoic acid, retinol, and 4-hydroxyphenylretinamide (nine studies), beta-carotene (seven studies), and calcium compounds (three studies). In addition, six promising new compounds are in Phase I or Phase II trials. These are: piroxicam, ibuprofen, oltipraz (a dithiolthione), difluoromethylornithine, glycyrrhetinic acid, and N-acetylcysteine. Key concepts related to the development of cancer chemopreventive agents are (1) the need for long-term administration, (2) the need for oral route of administration, (3) the matching of toxic side effects to degree of cancer risk.