José-Luis Giner

NOVEL STEROLS OF THE TOXIC DINOFLAGELLATE KARENIA BREVIS (DINOPHYCEAE): A DEFENSIVE FUNCTION FOR UNUSUAL MARINE STEROLS? 1

The “red tide” organism Karenia brevis (Davis) Hansen & Moestrup (=Gymnodinium breve Davis) produces a mixture of brevetoxins, potent neurotoxins responsible for neurotoxic shellfish poisoning in humans and massive fish kills in the Gulf of Mexico and the southern Atlantic coast of the United States. The sterol composition of K. brevis was found to be a mixture of six novel and rare Δ8(14) sterols. The two predominant sterols, (24R)‐4α‐methylergosta‐8(14), 22‐dienol and (24R)‐4α‐methyl‐27‐norergosta‐8(14), 22‐dienol, were named gymnodinosterol and brevesterol and represent potentially useful biomarkers for K. brevis. A possible function for such unusual marine sterols is proposed whereby structural modifications render the sterols non‐nutritious to marine invertebrates, reducing predation and thereby enhancing the ability of the dinoflagellates to form massive blooms.

Indirect Stimulation of Human Vγ2Vδ2 T Cells through Alterations in Isoprenoid Metabolism

Human Vγ2Vδ2 T cells monitor isoprenoid metabolism by recognizing (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), an intermediate in the 2-C-methyl-d-erythritol-4-phosphate pathway used by microbes, and isopentenyl pyrophosphate (IPP), an intermediate in the mevalonate pathway used by humans. Aminobisphosphonates and alkylamines indirectly stimulate Vγ2Vδ2 cells by inhibiting farnesyl diphosphate synthase (FDPS) in the mevalonate pathway, thereby increasing IPP/triphosphoric acid 1-adenosin-5′-yl ester 3-(3-methylbut-3-enyl) ester that directly stimulate. In this study, we further characterize stimulation by these compounds and define pathways used by new classes of compounds. Consistent with FDPS inhibition, stimulation of Vγ2Vδ2 cells by aminobisphosphonates and alkylamines was much more sensitive to statin inhibition than stimulation by prenyl pyrophosphates; however, the continuous presence of aminobisphosphonates was toxic for T cells and blocked their proliferation. Aminobisphosphonate stimulation was rapid and prolonged, independent of known Ag-presenting molecules, and resistant to fixation. New classes of stimulatory compounds—mevalonate, the alcohol of HMBPP, and alkenyl phosphonates—likely stimulate differently. Mevalonate, a rate-limiting metabolite, appears to enter cells to increase IPP levels, whereas the alcohol of HMBPP and alkenyl phosphonates are directly recognized. The critical chemical feature of bisphosphonates is the amino moiety, because its loss switched aminobisphosphonates to direct Ags. Transfection of APCs with small interfering RNA downregulating FDPS rendered them stimulatory for Vγ2Vδ2 cells and increased cellular IPP. Small interfering RNAs for isopentenyl diphosphate isomerase functioned similarly. Our results show that a variety of manipulations affecting isoprenoid metabolism lead to stimulation of Vγ2Vδ2 T cells and that pulsing aminobisphosphonates would be more effective for the ex vivo expansion of Vγ2Vδ2 T cells for adoptive cancer immunotherapy.

New and efficient synthetic routes to 1-deoxy-D-xylulose

Abstract The biochemically important deoxysugar 1-deoxy-D-xylulose was synthesized by improved methods. D-Tartaric acid is the starting material for a synthesis which proceeds via the intermediacy of 2,3,4-tribenzyl-D-threitol. Another, highly efficient route used the Sharpless asymmetric dihydroxylation of 5-benzyloxy-3-penten-2-one as its key step. These syntheses are especially useful for isotopic labeling.

The Pneumocystis carinii drug target S‐adenosyl‐L‐methionine:sterol C‐24 methyl transferase has a unique substrate preference

Pneumocystis is an opportunistic pathogen that can cause pneumonitis in immunodeficient people such as AIDS patients. Pneumocystis remains difficult to study in the absence of culture methods for luxuriant growth. Recombinant protein technology now makes it possible to avoid some major obstacles. The P. carinii expressed sequence tag (EST) database contains 11 entries of a sequence encoding a protein homologous to S‐adenosyl‐L‐methionine (SAM):C‐24 sterol methyl transferase (SMT), suggesting high activity of this enzyme in the organism. We sequenced the erg6 cDNA, identified the putative peptide motifs for the sterol and SAM binding sites in the deduced amino acid sequence and expressed the protein in Escherichia coli. Unlike SAM:SMT from other organisms, the P. carinii enzyme had higher affinities for lanosterol and 24‐methylenelanosterol than for zymosterol, the preferred substrate in other fungi. Cycloartenol was not a productive substrate. With lanosterol and 24‐methylenelanosterol as substrates, the major reaction products were 24‐methylenelanosterol and pneumocysterol respectively. Thus, the P. carinii SAM:SMT catalysed the transfer of both the first and the second methyl groups to the sterol C‐24 position, and the substrate preference was found to be a unique property of the P. carinii SAM:SMT. These observations, together with the absence of SAM:SMT among mammals, further support the identification of sterol C‐24 alkylation reactions as excellent targets for the development of drugs specifically directed against this pathogen.

Sterols of the brown tide alga Aureococcus anophagefferens

Abstract Aureococcus anophagefferens , an alga responsible for “brown tide”, contains 24-methylenecholesterol as 48% of the free sterols and ( E )- and ( Z )-24-propylidenecholesterol as 32% and 12% respectively. This is the first isolation of appreciable amounts of ( Z )-24-propylidenecholesterol from an alga and suggests that Aureococcus anophagefferens may be the source of the ( Z )-24-propylidenecholesterol isolated from various marine invertebrates. 24-Propylidenecholesterol is proposed to be a chemotaxonomic marker for the Pelagophyceae, a new class of chrysophytes which includes the Sarcinochrysidales.

Sterols and fatty acids of three harmful algae previously assigned as Chattonella.

Sterol and fatty acid compositions were determined for three harmful algal species previously classified in the genus Chattonella (Raphidophyceae): the new genus Chloromorum toxicum (ex Chattonella cf. verruculosa), Verrucophora farcimen (Dictyochophyceae), previously Chattonella aff. verruculosa, and Verrucophora verruculosa (=Pseudochattonella verruculosa) previously Chattonella verruculosa. The major fatty acids of C. toxicum were 14:0, 16:0, 18:1n-9, 18:4n-3 and 20:5n-3, and those of the Verrucophora strains were. 14:0, 16:0, 18:0, 18:4n-3, 18:5n-3 and 22:6n-3. C. toxicum contained the 24beta-ethyl sterols, poriferasterol and clionasterol, as its major sterols. For comparison, the stereochemistry of the 24-ethyl sterols of two raphidophytes, Chattonella marina and Heterosigma akashiwo, was determined to be 24alpha and 24beta, respectively. Both Verrucophora strains contained the 27-nor sterol occelasterol as the only detected sterol. This was the first time occelasterol has been found in algae.

Sterol Chemotaxonomy of Marine Pelagophyte Algae

Several marine algae of the class Pelagophyceae produce the unusual marine sterol 24‐propylidenecholesterol, mainly as the (24E)‐isomer. The (24Z)‐isomer had previously been considered as a specific biomarker for Aureococcus anophagefferens, the ‘brown tide’ alga of the Northeast coast of the USA. To test this hypothesis and to generate chemotaxonomic information, the sterol compositions of 42 strains of pelagophyte algae including 17 strains of Aureococcus anophagefferens were determined by GC analysis. A more comprehensive sterol analysis by HPLC and 1H‐NMR was obtained for 17 selected pelagophyte strains. All strains analyzed contained 24‐propylidenecholesterol. In all strains belonging to the order Sarcinochrysidales, this sterol was found only as the (E)‐isomer, while all strains in the order Pelagomonadales contained the (Z)‐isomer, either alone or together with the (E)‐isomer. The occurrence of Δ22 and 24α‐sterols was limited to the Sarcinochrysidales. The first occurrence of Δ22‐24‐propylcholesterol in an alga, CCMP 1410, was reported. Traces of the rare sterol 26,26‐dimethyl‐24‐methylenecholesterol were detected in Aureococcus anophagefferens, and the (25R)‐configuration was proposed, based on biosynthetic considerations. Traces of a novel sterol, 24‐propylidenecholesta‐5,25‐dien‐3β‐ol, were detected in several species.

A reinvestigation of the biosynthesis of lanosterol in Euphorbia lathyris

Abstract The latex of Euphorbia lathyris was shown to be capable of producing lanosterol, as well as cycloartenol, butyrospermol and hopenol B, via the enzymatic cyclization of 2,3-oxidosqualene. The conversion of cycloartenol to lanosterol could not be demonstrated using the same latex. The triterpene composition of the latex of Euphorbia lathyris is reported.

Dinoflagellates cyclize squalene oxide to lanosterol

Abstract Direct enzymatic cyclization of squalene oxide to cycloartenol has now been demonstrated in eight algal phyla, whereas five species of dinoflagellates produced lanosterol. This discovery of a lanosterol pathway in dinoflagellates is of clear taxonomic significance. In addition, these findings represent evidence against an assumed biochemical linkage between photosynthesis and cycloartenol production from squalene oxide.

Stereospecific Synthesis of 24-Propylcholesterol Isolated from the Texas Brown Tide

Abstract The alga that causes the ‘Texas brown tide’, Aureoumbra lagunensis, contains 24-propylcholesterol, a potentially useful biomarker for this organism. The stereochemical configuration at C-24 was determined through synthesis using the Johnson orthoester Claisen rearrangement. Both (24R)- and (24S)-24-propylcholesterol, as well as (24R)- and (24S)-Δ22 24-propylcholesterol, were synthesized and characterized. The naturally occurring isomer was found to be (24R)-24-propylcholesterol.