Clinical and molecular heterogeneity of phenylalanine hydroxylase deficiencies in France

RFLPs of 68 normal and 74 mutant alleles at the phenylalanine hydroxylase (PAH) locus were determined in 37 French kindreds. A total of 23 haplotypes, including 18 normal and 16 mutant alleles, were observed. Two-thirds of all mutant alleles were confined within only four haplotypes, while the last third was accounted for by 12 haplotypes, including eight haplotypes absent from Caucasian pedigrees reported thus far. Several mutant haplotypes were present in typical phenylketonuria only, others were present in variants only, and some were present in both. In addition, a particular mutant haplotype (haplotype 2) was found to harbor different mutations in our series, resulting in either typical phenylketonuria or in mild hyperphenylalaninemias. The diploid combination of so many mutant haplotypes in PAH-deficient patients and of compound heterozygosity at the PAH locus in southern Europe might account for the broad spectrum of individual phenotypes observed in France.     

Cell surface properties correlated with cohesion in Myxococcus xanthus.

The gliding behavior of Myxococcus xanthus cells is controlled by two multigene systems, A and S, which encode information for adventurous and social behaviors, respectively. The S system can be genetically disrupted through mutation, such as a dsp mutation, or phenotypically disrupted by treating cells with the diazo dye Congo red (Arnold and Shimkets, J. Bacteriol. 170:5765-5770, 1988). One of the functions controlled by the S system is cell agglutination. Immediately after the induction of agglutination, wild-type cells begin to form aggregates, and within 30 min the cells are packed side-to-side in clumps containing thousands of cells. Changes in the cohesive properties of S+ cells are correlated with changes in the topology of the cell surface observed by electron microscopy. Two types of cell-associated appendages were observed on wild-type cells: thin filaments (ca. 5 nm in diameter), which have been called fimbriae or pili, at one cell pole, and thick, flaccid filaments (ca. 50 nm in diameter), referred to as fibrils, at both the sides and tips of cells. Cohesion was correlated with the secretion of the thick fibrils, which coat the cell surface and form an extracellular matrix in which the cells are interconnected. Several lines of evidence suggest that these thick fibrils are involved in cohesion. First, Dsp cells were unable to agglutinate or secrete this extracellular material. Second, wild-type cells which were treated with Congo red neither agglutinated nor secreted the extracellular fibrils. Finally, removal of the Congo red from wild-type cells restored cohesion and also restored production of the thick fibrils. Attempts to estimate the efficiency with which two cells cohered following collision suggested that under optimal conditions, one in three collisions resulted in stable contact. The collision efficiency decreased linearly as the cell density increased, suggesting a cell density-dependent regulation of cohesion. Some aspects of gliding behavior can be explained in terms of an inducer and an inhibitor of S motility.     

The comparative influence of substituted phenols (especially chlorophenols) on yeast cells assayed by electro-rotation and other methods

The toxicity of 31 phenols was studied by electro-rotation of yeast cells. Control yeast cells show both anti-field and co-field rotation, depending upon the field frequency applied. After treatment with supra-threshold amounts of phenols the anti-field rotation is weakened or abolished and a stronger co-field rotation can be seen. The proportion of cells showing the co-field rotation was found to be a sensitive measure of toxicity. Doses of 2.2 mumol/l of pentachlorophenol, or of 0.3 mumol/l of pentabromophenol were detectable after 3 h incubation at pH 4.0. At a given pH, the toxicity of the chlorophenols correlated extremely well with their octanol:water partition coefficients (Pow). The complete set of phenols showed fair overall correlation with Pow, but less good correlation with their acidity constants (pKa). In particular the toxicity of a given phenol was less than predicted from its pKa if the incubation pH was higher than the pKa. Biochemical assays on 23 of the phenols showed that the rotational sensitivity runs closely parallel to the sensitivities of cell growth rate and of the plasmamembrane ATPase, but less closely to the inhibition of purine incorporation. It appears that the electro-rotation method provides a useful and rapid test for the presence of organic ecotoxins. The test enables us to distinguish differences between single cells, and is comparable in sensitivity to biochemical tests that use vesicles or homogenates derived from a cell population.     

The rate of lateral diffusion of phospholipids in erythrocyte microvesicles

31P-NMR spectra of phospholipids in membranes of erythrocyte microvesicles isolated from outdated blood units were recorded in the temperature range 5 to 55 degrees C. Within that range the lineshape is strongly influenced by an increasing rate of lateral diffusion of phospholipids. At 36 degrees C a diffusion constant, D, of (2 +/- 1) X 10(-12) m2/s was obtained. The diffusion rate is by a factor of 3 to 10 greater than in erythrocyte membranes measured by the photobleaching technique and is comparable with values obtained for several lipid model membranes. The differences in lateral diffusion rates are probably connected with the depletion of microvesicle membranes in membrane proteins.     

The heterochromatin of grasshoppers from the Caledia captiva species complex

A highly repeated family of sequences from the grasshopper Caledia captiva shows a dispersed distribution at the cytological level. Members of this 185 bp sequence family are not restricted to C-band heterochromatin, but rather are distributed in regions which appear as euchromatin in C-banded chromosomes. Sequence variation in this family is equivalent (14%–16%) at all levels of taxonomic comparison from within a population to between species. However, contiguous repeats demonstrate a much lower level of variation (9%). These, and other data, indicate that the concept of sequence homogeneity within a family of highly repeated sequences must be qualified with respect to the extensive variation between members of a given family. Comparison of the data for the 185 bp family with those from a study of a second highly repeated family, from the same taxon, demonstrates divergent patterns of evolution. Thus, the 185 bp repeats show much greater sequence variation, as well as a seemingly random pattern of incorporation of base pair alterations. The factors which may contribute to the observed pattern of variation include the time since the sequence family originated, its cytological distribution, the frequency of unequal crossing over and gene conversion and natural selection.     

Control of rat gastric somatostatin release by gamma-aminobutyric acid (GABA)

The influence of gamma-aminobutyric acid (GABA) on gastric somatostatin and gastrin release was studied using an isolated perfused rat stomach preparation. GABA dose-dependently inhibited somatostatin release (maximal inhibition of 44% at 10(-5)M GABA), whereas gastrin secretion was not affected. The GABA agonist muscimol led to a decrease in somatostatin release of similar magnitude. The GABA-induced changes were partially reversed by 10(-5)M atropine. Gastrin secretion was not influenced by either protocol. It is concluded that GABA as a putative neurotransmitter in the enteric nervous system is inhibitory to rat gastric somatostatin release in vitro via cholinergic pathways.     

A new method for the reconstitution of the anion transport system of the human erythrocyte membrane

Summary The anion transport protein of the human erythrocyte membrane, band 3, was solubilized and purified in solutions of the non-ionic detergent Triton X-100. It was incorporated into spherical lipid bilayers by the following procedure: (1) Dry phosphatidylcholine was suspended in the protein solution. Octylglucopyranoside was added until the milky suspension became clear. (2) The sample was dialyzed overnight against detergentfree buffer. (3) Residual Triton X-100 was removed from the opalescent vesicle suspension by sucrose density gradient centrifugation and subsequent dialysis. Sulfate efflux from the vesicles was studied, under exchange conditions, using a filtration method. Three vesicle subpopulations could be distinguished by analyzing the time course of the efflux. One was nearly impermeable to sulfate, and efflux from another was due to leaks. The largest subpopulation, however, showed transport characteristics very similar to those of the anion transport system of the intact erythrocyte membrane: transport numbers (at 30°C) close to 20 sulfate molecules per band 3 and min, an activation energy of approx. 140 kJ/mol, a pH maximum at pH 6.2, saturation of the sulfate flux at sulfate concentrations around 100mm, inhibition of the flux by H₂DIDS and flufenamate (approx.K _l-values at 30°C: 0.1 and 0.7 m, respectively), and right-side-out orientation of the transport protein (as judged from the inhibition of sulfate efflux by up to 98% by externally added H₂DIDS). Thus, the system represents, for the first time, a reconstitution of all the major properties of the sulfate transport across the erythrocyte membrane.     

The stereochemical course of phosphoryl transfer catalyzed by herpes simplex virus type I-induced thymidine kinase

Herpes simplex virus type I (HSV-I)-induced thymidine kinase has been shown to catalyze phosphoryl transfer from adenosine 5'-[gamma-(S)-16O,17O,18O]triphosphate to thymidine with inversion of configuration at phosphorus. The simplest interpretation of this result is that phosphoryl transfer occurs by a single in-line group transfer between ATP and thymidine within the ternary enzyme complex.     

Analogs of diaminopimelic acid as inhibitors of meso-diaminopimelate decarboxylase from Bacillus sphaericus and wheat germ

Analogs (1----6) of diaminopimelic acid have been synthesized and tested for inhibition of meso-diaminopimelate decarboxylases from Bacillus sphaericus IFO 3525 and from wheat germ (Triticum vulgaris). Difluoromethyl diaminopimelate 1 does not irreversibly inactivate or strongly competitively inhibit either enzyme. Lanthionine sulfoxides (2ab, 2c, and 2d) are good competitive inhibitors (about 50% inhibition at 1 mM) of both decarboxylases. The meso and LL-isomers of lanthionine sulfone (3ab and 3c) and lanthionine (6ab and 6c) are weaker competitive inhibitors (about 50% inhibition at 10-20 mM). The corresponding DD-isomers (3d and 6d) are less effective. The N-modified analogs are the most potent competitive inhibitors. The inhibition constant (Ki) values for B. sphaericus and wheat germ decarboxylases with N-hydroxydiaminopimelate 4 (mixture of isomers) are 0.91 and 0.71 mM, respectively; for the N-aminodiaminopimelate 5 (mixture of isomers) the Ki values are 0.10 and 0.084 mM, respectively. These N-modified analogs do not effectively inhibit L-lysine decarboxylase. None of the compounds showed any time-dependent inactivation of the decarboxylases, in contrast to behavior of other pyridoxal phosphate-dependent enzymes with analogous substrate derivatives. Possible mechanisms of inhibition are discussed. In preliminary tests for antibiotic activity 4 and 5 both gave 75% growth inhibition of Bacillus megaterium at 20 micrograms/ml in defined media. Other analogs (1----3) showed essentially no antibacterial activity.     

Phosphatidyl glycerolphosphate serves as glycerolphosphate donor in polymer synthesis

Phosphatidyl glycerolphosphate was found to serve as the glycerolphosphate donor for polymer synthesis. When CDP-diglyceride and radiolabeled glycerolphosphate were incubated with the membrane enzyme prepared from Streptococcus sanguis, active syntheses of radiolabeled lipids and polymers were observed. The synthesis of polymer was not inhibited by low concentration of unlabeled phosphatidylglycerol. When [3H, 32P]glycerolphosphate was used, the polymer synthesized contained both 3H and 32P. The lipids formed were characterized as phosphatidylglycerol and phosphatidyl glycerolphosphate. The polymers formed from the latter were characterized as lipoteichoic acid like compounds by sodium dodecylsulfate-polyacrylamide gel electrophoresis.