W. Dierick

Identification and characterization of dolichyl dolichoate, a novel isoprenoic derivative in bovine thyroid.

Inspection of the TLC pattern of the neutral lipid fraction of bovine thyroid reveals, in addition to cholesteryl esters and dolichyl fatty acid esters, the presence of a not yet identified compound in the most apolar lipid region. This unknown compound was purified on a preparative scale by silicic acid column chromatography, Lipidex-5000 gel-filtration chromatography and preparative thin-layer chromatography. By chemical (hydrolysis and reduction) and spectroscopic (ultraviolet, infrared, NMR, mass spectroscopy) methods this lipid was identified as dolichyl dolichoate. The homologue pattern was analyzed, by HPLC and FDMS, respectively.

Two cases of mucopolysaccharidosis type III (Sanfilippo)

SummaryAnatomopathological studies of one case of Sanfilippo disease types A and C, respectively, are presented. The storage phenomenon is very severe in the central as well as in the peripheral nervous system of both patients. Light microscopy does not show significant differences between the two cases. Electron microscopy of the nervous system reveals in both cases the presence of variable amounts of zebra bodies and of membrano-granulo-vacuolar inclusions. The presence of larger amounts of zebra bodies in the type A case and of larger quantities of membrano-granulo-vacuolar inclusions in the type C case constitute probably a non-distinctive feature between the two types. Light and electron microscopic studies of visceral organs do not disclose significant differences either. It is concluded that no major morphological differences between Sanfilippo disease types A and C can be observed.

Multiple system atrophies. A neuropathological and neurochemical study.

Abstract The 5 familial cases which are reported here, showed a similar clinical course characterized by an early onset and a very long duration provided adequate nursing care was available. The main features were a very prominent pyramidal syndrome, less pronounced or partially masked cerebellar, myoclonic and extrapyramidal syndromes and preterminal sphincter disturbances which accounted for feeding difficulties, malnutrition and infections. Epilepsy was not infrequent but its management was possible with modest does of antiepileptic drugs. Investigations during life are helpful in ruling out some lipidoses such as metachromatic leucodystrophy, but they did not give any useful evidence as to pathogenesis. If one accepts an autosomal recessive inheritance (this is not completely certain), it is clear that neither clinical examination nor investigations are at present helpful in the detection of heterozygotes. The main pathological findings were multiple system atrophies and leucodystrophy. The system atrophies were qualitatively similar to those found in the classical spinocerebellar degenerations but they were much more widespread above the spinal level. The constellation of optic, thalamic, pontine, olivary, pyramidal and other long-tract lesions, among which the thalamic degeneration was unusually pronounced, appears to be unique among the abiotrophic processes. The second major pathological feature which contributes to the specificity of the syndrome, is the diffuse sclerosis of the white matter. If one attempts to classify these lesions into the conventional group of leucodystrophies, then they belong to the very large and ill-defined group of orthochromatic leucodystrophies (for a review, see Peiffer 1970b). Lipid analyses of postmortem brain tissue in 2 patients revealed a decrease in the lipid content of the white matter. No such effect was noted in grey matter. This drastic reduction - true for neutral lipids, glycolipids and phospholipids - could be interpreted as the chemical manifestation of demyelination. HFA deficiency occurring both in white and grey matter was the striking biochemical feature in our patients. This finding is rather unique and could perhaps be regarded as a primary event in this disease. A deficiency of the α-hydroxylating system is the most likely cause of this familial disorder.

Nucleotide sequence analysis of the CT operon of the Vibrio cholerae classical strain 569B.

The complete nucleotide sequence of the Vibrio cholerae classical strain 569B was determined. The results prove the exactness of the amino acid CT B sequence published by Takao et al. (1985, Eur. J. Biochem. 146, 503-508). A comparison is made with already reported CT genes.

Structural features of the binding site of cholera toxin inferred from fluorescence measurements.

The dependence on pH of the fluorescence of cholera toxin and its A and B subunits has been studied at 25 degrees C. The fluorescence intensity of cholera toxin is highly pH-dependent. In the pH range 7-9.5 it reaches a maximum corresponding to a quantum yield of 0.076. In the pH range 4-7 a strong increase in fluorescence intensity is observed (delta Q/Qmax = 0.64). Evaluation of the pH sensitivity of the fluorescence intensity of the A and B subunits reveals that the B subunit is mainly responsible for the observed pH effect (delta Q/Qmax for B subunit = 0.64). The intensity changes are paralleled by similar although less pronounced changes in the average fluorescence excited state life-time tau (delta tau/tau max = 0.33 for cholera toxin). Fluorimetric titration of the B subunit, which is related to the indole fluorescence of the lone Trp-88, reveals that the fluorescence intensity changes in the pH range 4-7 are due to reaction of two types of ionizable quencher displaying apparent pKa values of 4.4 and 6.2, respectively. It is suggested that the increase in fluorescence intensity with a midpoint at pH 6.2 is the result of deionization of the imidazolium side-chain of one or two out of the four histidine residues present in each beta-polypeptide chain, whereas a deionized carboxyl group is responsible for the quenching with midpoint at pH 4.4. Complex formation of cholera toxin or B subunit with the monosialoganglioside GM1 or the oligosaccharide moiety of GM1 (oligo-GM1) completely prevents the quenching by both quenchers. Addition of 6 M urea also eliminates the pH effect. The quenching is not the result of the dissociation of the B subunit into its constituent monomers. Upon fluorimetric titration of cholera toxin or B subunit above pH 9, a progressive drop in both fluorescence intensity and tau occurs. This decrease could be due to energy transfer from the indole moiety of Trp-88 to ionized tyrosines or by quenching through an unprotonated epsilon-amino group of lysine. Fluorimetric titration of the A subunit indicates that the tryptophan fluorescence is only moderately altered by ionizable groups displaying a pKa in the range 4 to 9. Activation of A subunit does not affect this lack of pH sensitivity. Above pH 9, however, a much more significant drop in the fluorescence intensity of activated A subunit occurs. The structural implications of the results are discussed.

Two cases of mucopolysaccharidosis type-III (Sanfilippo) : a biochemical-study

The mucopolysaccharide and lipid composition of human nervous tissue and viscera from one case of Sanfilippo disease type A and one case of Sanfilippo disease type C, were investigated. In the brain a moderate increase of acid glycosaminoglycans occurred. This phenomenon was much more pronounced in the viscera, especially in the liver. In all tissues this increase was mainly due to an accumulation of heparan sulphate. Changes in lipid composition were noted, but can be regarded as secondary effects. The biochemical results reported also suggest some general conclusions. (a) AGAG and lipid analyses do not permit differentiation between the subtypes of Sanfilippo disease. (b) The differences in lipid composition can probably be considered as consequences of variation in secondary effects. (c) The severe demyelination in brain correlates well with the biochemical lipid analysis. However, in other instances it remains difficult to bridge the gap still existing between some morphological and biochemical data.

Characterization of the in vitro conversion of dolichol to dolichoate in bovine thyroid.

The enzymic conversion of dolichol into dolichoic acid has been studied in bovine thyroid subcellular fractions using [1-3H]dolichol as a substrate. The presence of conversion activity could be demonstrated in both the mitochondrial- and supernatant fractions. Investigation of cofactor requirements revealed that NAD+ was essential for reaching optimal activity. From kinetic studies Km-values of 3.5-4 microM and 0.29 mM could be calculated for, respectively, dolichol and NAD+ using the mitochondrial fraction as an enzyme source. No inhibitory effects from ethanol or pyrazole were detected suggesting that alcohol dehydrogenase is not involved in the dolichol-->dolichoate conversion as observed in a bovine thyroid mitochondrial fraction. From inhibitor studies the conversion system behaves distinctly differently from the NADP(+)-depending microsomal oxidoreductase as well as from catalase. The conversion activity in the supernatant on the other hand must be ascribed, at least partially, to a side activity of alcohol dehydrogenase.

Modification of the adenylate cyclase activity of bovine thyroid plasma membranes by manipulating the ganglioside composition with a nonspecific lipid transfer protein

Gangliosides (GM1, GT1b, GD3) were incorporated in bovine thyroid plasma membranes using the nonspecific lipid transfer protein from beef liver. The transfer of GT1b or GD3 in the presence of 16 units of transfer protein was twice as high as that of GM1. However, taking into account the spontaneous exchange (approximately 8% for GT1b or GD3 and 1% for GM1) the transfer protein seemed to be more effective for GM1. Incorporation of these gangliosides in bovine thyroid plasma membranes caused a concentration dependent inhibition of the TSH-stimulated adenylate cyclase activity. The forskolin-stimulated adenylate cyclase activity was not significantly affected by ganglioside modification of the plasma membranes, indicating that the gangliosides do not act at the level of the catalyst of adenylate cyclase. Binding experiments on the other hand revealed that TSH binding to bovine thyroid plasma membranes was inhibited with the same order of efficacy (GT1b greater than GD3 greater than GM1) and to the same extent as their inhibitory effect on TSH stimulation. Therefore, this indicates that the ganglioside induced drop in TSH binding might be an important factor in the decrease in TSH-stimulated adenylate cyclase activity. Incorporation of GT1b or GD3 (approximately 11 nmol) in bovine thyroid plasma membranes, however, also induced a substantial decrease in cholera toxin-stimulated adenylate cyclase activity (approximately 30%) and to a lesser degree a decrease in NaF-stimulated activity (approximately 17%), whereas GM1 incorporation did not significantly affect these stimulated activities. These latter inhibitory effects were paralleled by changes in fluorescence steady-state anisotropy: GT1b modification of the plasma membranes provoked a slight increase in TMA-DPH anisotropy, whereas the anisotropy of DPH was substantially enhanced after incorporation of GD3 or GT1b. These results suggest that gangliosides might also interfere with the coupling between the alpha-subunit of the stimulatory GTP-binding regulatory protein and the catalyst of the adenylate cyclase system by affecting the membrane fluidity.

Interaction of a cholera toxin derivative containing a reduced number of receptor binding sites with intact cells in culture

Hybrid CTB (hCTB), having only one or two functional binding sites, has been constructed from two chemically inactivated derivatives of CTB. One inactive derivative consisted of CTB formylated in the lone Trp-88 of each beta-chain (fCTB), whereas the other inactive derivative consisted of CTB specifically succinylated in three amino groups located in or near the receptor binding site (sssCTB). hCTB, fCTB and sssCTB were able to reassociate with CTA and form the corresponding holotoxins hCT, fCT and sssCT as measured by gel filtration chromatography. In contrast to fCT and sssCT, hCT could increase the cAMP content of intact Vero cells in a time- and dose-dependent way: concentrations as low as a few nanograms of hCT per milliliter caused a significant increase in the intracellular cAMP level. The maximal cAMP level induced by hCT (1 microgram/ml) was, however, more than 2-fold lower than that elicited by its native counterpart. At saturating ligand concentrations and at 37 degrees C, the lag periods and rates of CT and hCT induced cAMP accumulation were essentially the same. Treatment of Vero and HeLa cells with GM1 did not affect their difference in response to CT and hCT. When Vero cells treated with hCT were incubated for longer periods of time, a further slow accumulation of cAMP occurred until after about 20 h cAMP levels of cells exposed to CT or hCT were essentially the same. In contrast to Vero and HeLa cells, human skin fibroblasts exhibited an almost identical response to CT as well as to hCT. Acidotropic agents such as chloroquine and monensin affected the CT and hCT induced increase in cAMP content of Vero cells, fibroblasts and GM1 treated Hela cells in a similar way. The results are consistent with the view that CT receptor recognition domains are shared between adjacent beta-chains, that pentavalent binding appears not to be essential for cytotoxicity and that in the cell types studied intracellular processing of CT, hCT is involved.

Intracellular and Extracellular Flow of Dolichol

Dolichols belong to a family of polymeric lipids with the isoprene unit as the repeating building block. This lipid class represents a relative large group of natural products displaying a wide variety of biological functions (Table I) (Rip et al., 1985). The existence of dolichols was first reported by Hemming and coworkers (Pennock et al., 1960; Burgos et al., 1963).