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.     

Capitalist Philanthropy and Russian Revolutionaries: The Jesup North Pacific Expedition (1897-1902)

This review of the Jesup North Pacific Expedition, still the most important expedition in American anthropology, gives an idea of the goals and hazards of fieldwork around 1900, the pitfalls of international research, the tensions between anthropologists and host populations, the careers of early anthropologists, the role of private philanthropy, and the character of anthropology at the turn of the century. Franz Boas was the Expedition's linchpin. His organization of the Expedition, the way he handled problems, and his personal concerns reveal aspects of his view of anthropology and some of his basic attitudes.     

Solubilization and reconstitution of the formylmethionylleucylphenylalanine receptor coupled to guanine nucleotide regulatory protein

We describe the solubilization, resolution, and reconstitution of the formylmethionylleucylphenylalanine (fMet-Leu-Phe) receptor and guanine nucleotide regulatory proteins (G-proteins). The receptor was solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. Guanine nucleotides decreased the number of high-affinity binding sites and accelerated the rate of dissociation of the receptor-ligand complex, suggesting that the solubilized receptor remained coupled to endogenous G-proteins. The solubilized receptor was resolved from endogenous G-proteins by fractionation on a wheat germ agglutinin (WGA)-Sepharose 4B column. High-affinity [3H]fMet-Leu-Phe binding to the WGA-purified receptor was diminished and exhibited reduced guanine nucleotide sensitivity. High-affinity [3H]fMet-Leu-Phe binding and guanine nucleotide sensitivity were reconstituted upon the addition of purified brain G-proteins. Similar results were obtained when the receptor was reconstituted with brain G-proteins into phospholipid vesicles by gel filtration chromatography. In addition, we also demonstrated fMet-Leu-Phe-dependent GTP hydrolysis in the reconstituted vesicles. The results of this work indicate that coupling of the fMet-Leu-Phe receptor to G-proteins converts the receptor to a high-affinity binding state and that agonist produces activation of G-proteins. The resolution and functional reconstitution of this receptor should provide an important step toward the elucidation of the molecular mechanism of the fMet-Leu-Phe transduction system in neutrophils.     

Genetic Controls over Activities of Tyrosinase and Dopachrome Conversion Factor in Murine Melanocytes

We evaluated the three catalytic activities of tyrosinase and one activity of dopachrome conversion factor (DCF) in extracts made from skins of 6-day-old yellow and nonyellow mice. At least one of the catalytic activities of tyrosinase and of DCF correlate with the color of pigment being produced in the hair follicles of the mice. We use these data to evaluate existing hypotheses about the mechanism of the interacting genetic controls over melanogenesis.     

Cellular Localization of the Molecular Forms of Acetylcholinesterase in Primary Cultures of Rat Sympathetic Neurons and Analysis of the Secreted Enzyme

The secretion and cellular localization of the molecular forms of acetylcholinesterase (AChE) were studied in primary cultures of rat sympathetic neurons. When cultured under conditions favoring a noradrenergic phenotype, these neurons synthesized and secreted large quantities of the tetrameric G4, and the dodecameric A12 forms, and minor amounts of the G1 and G2 forms. When these neurons adopted the cholinergic phenotype, i.e., in the presence of muscle-conditioned medium, the development of the cellular A12 form was completely inhibited. These neurons secreted only globular, mainly G4, AChE. Both cellular and secreted A12 AChE in adrenergic cultures aggregated at an ionic strength similar to that of the culture medium, raising the hypothesis that this form was associated with a polyanionic component of basal lamina. In noradrenergic neurons, 60-80% of the catalytic sites were exposed at the cell surface. In particular, 80% of G4 form, but only 60% of the A12 form, was external, demonstrating for the A12 form a sizeable intracellular pool. The hydrophobic character of the molecular forms was studied in relation to their cellular localization. As in muscle cells, most of the G4 form was membrane-bound. Whereas 76% of the cell surface A12 form was solubilized in the aqueous phase by high salt concentrations, only 50% of the intracellular A12 form was solubilized under these conditions. The rest of intracellular A12 could be solubilized by detergents and was thus either membrane-bound or entrapped in vesicles originating from, e.g., the Golgi apparatus.     

Dopamine Metabolism in Hypoxanthine-Guanine Phosphoribosyltransferase-Deficient Variants of PC12 Cells

Lesch-Nyhan syndrome results from a deficiency of hypoxanthine-guanine phosphoribosyltransferase (HPRT). It is manifest by behavioral abnormalities, including self-mutilation, and evidence of abnormal 3,4-dihydroxyphenylethylamine (dopamine) metabolism. To assess whether an HPRT deficiency in a dopaminergic cell can adversely affect dopamine metabolism in that cell, dopamine metabolism was examined in HPRT-deficient variants of PC12 pheochromocytoma cells and in cells that had regained HPRT activity by virtue of transformation with a recombinant retrovirus containing the human gene for HPRT. There was no correlation between HPRT activity and endogenous dopamine levels, dopamine uptake, dopamine release, or monoamine oxidase activity. Transformation with the HPRT retrovirus did not adversely affect dopamine metabolism.     

Reaction pathway of bovine aortic lysyl oxidase

The catalysis of amine oxidation by lysyl oxidase has been probed to assess for the likely order of substrate binding and product release and to discriminate between mechanistic alternatives previously proposed for other copper-dependent amine oxidases using molecular oxygen as a substrate. Lineweaver-Burk plots revealed a pattern of parallel lines when the oxidation of n-butylamine was followed at different fixed concentrations of oxygen consistent with a "ping-pong" kinetic mechanism in which the aldehyde is produced and released before the binding of oxygen, the second substrate. Initial burst experiments revealed the ability of lysyl oxidase to form and release n-butyraldehyde in amounts stoichiometric with functional active site content in the absence of oxygen, consistent with the ping-pong kinetics obtained. Reciprocal plots of n-butylamine oxidation in the presence of fixed concentrations of the reaction products were consistent with a Uni Uni Uni Bi ping-pong kinetic mechanism with the aldehyde being the first, H2O2 the second, and ammonia the last departing product. Moreover, spectral studies of the oxidation of p-hydroxybenzylamine by lysyl oxidase indicated that the enzyme does not process the amine substrate to a noncovalently bound p-hydroxybenzaldimine intermediate subsequently to be hydrolyzed to p-hydroxybenzaldehyde. The kinetic mechanism of lysyl oxidase thus appears to be similar to those described for diamine oxidase and pig plasma monoamine oxidase.     

Colorimetric plasma assay for the bentiromide test (BT-PABA) for exocrine pancreatic insufficiency

Bentiromide is a synthetic peptide, N-benzoyl-L-tyrosyl-p-aminobenzoic acid, which has been used as a test for exocrine pancreatic function. Following oral administration, bentiromide is hydrolyzed by chymotrypsin to yield free p-aminobenzoic acid (PABA) which is absorbed, conjugated and excreted in the urine. The PABA conjugates reach their peak levels in blood in 90-120 min. Healthy individuals have higher levels of PABA than patients with pancreatic insufficiency. A simple, accurate, and precise method for the determination of PABA in blood has been developed and validated. The plasma (1 ml) is deproteinized by perchloric acid. The conjugates are hydrolyzed and the total PABA is determined colorimetrically by the Bratton-Marshall test. The standard curve in plasma is linear up to 8 micrograms/ml of PABA. A similar semimicro method using 200 microliter of plasma suitable for pediatric samples shows comparable results. Average analytical recovery is 97% and precision studies of pooled within-run and total between-run showed CV% of 5.0 and 5.7%, respectively.     

Permeability of the squid giant axon to organic cations and small nonelectrolytes

Summary The permeability of the Na channel of squid giant axon to organic cations and small nonelectrolytes was studied. The compounds tested were guanidinium, formamidinium, and¹⁴C-labeled urea, formamide, thiourea, and acetone. Permeability was calculated from measurements of reversal potential and influx on internally perfused, voltage clamped squid axons. The project had two objectives: (1) to determine whether different methods of measuring the permeability of organic cations yield similar values and (2) to see whether neutral analogs of the organic cations can permeate the Na channel. Our results show that the permeability ratio of sodium to a test ion depends upon the ionic composition of the solution used. This finding is consistent with the view put forward previously that the Na channel can contain more than one ion at a time. In addition, we found that the uncharged analogs of permeant cations are not measurably permeant through the Na channel, but instead probably pass through the lipid bilayer.