Characterization of mesenchymal progenitor cell populations from non-epithelial oral mucosa.

OBJECTIVES: The characteristics of cell populations extracted from oral mucosal non-epithelial tissues and their ability to differentiate were evaluated in vitro as a potential source of cells for mandibular and corneal regeneration. MATERIALS AND METHODS: Oral mucosal non-epithelial cells (OMNECs) were extracted from tissue samples and were studied by flow cytometry and RT-PCR. Cells differentiating into osteoblasts, adipocytes, chondrocytes, neurocytes, or keratocytes were characterized by RT-PCR and cell staining. RESULTS: OMNECs expressed CD44, CD90, CD105, CD166, and STRO-1 antigens, which are markers for mesenchymal stem cells. In addition, Oct3/4, c-Myc, Nanog, KLF4, and Rex, which are expressed by embryonic or pluripotent stem cells, were detected by RT-PCR. Expression of CD49d, CD56, and PDGFRα, proteins closely associated with the neural crest, was observed in OMNECs, as was expression of Twist1, Sox9, Snail1 and Snail2, which are early neural crest and neural markers. Specific differentiation markers were expressed in OMNECs after differentiation into osteoblasts, adipocytes, chondrocytes, or keratocytes. CONCLUSIONS: Populations of OMNECs may contain both mesenchymal stem cells and neural crest origin cells and are a potential cell source for autologous regeneration of mandibular or corneal stroma.

Rhodovulum mangrovi sp. nov., a phototrophic alphaproteobacterium isolated from a mangrove forest sediment sample.

A novel Gram-staining-negative, purple non-sulfur bacterium, strain AK41(T), was isolated from a sediment sample collected from Coringa mangrove forest, Andhra Pradesh, India. A red-brownish-coloured culture was obtained on modified Pfennig medium after enrichment with 2 % NaCl and 0.3 % pyruvate under 2000 lx illumination. Individual cells were ovoid-rod-shaped and non-motile. Bacteriochlorophyll a and carotenoids of the spheroidene series were present as photosynthetic pigments. Strain AK41(T) was halophilic and grew photoheterotrophically with a number of organic compounds as carbon sources and electron donors. It was unable to grow photoautotrophically. It did not utilize sulfide or thiosulfate as electron donors. The fatty acids were found to be dominated by C16 : 0 and C18 : 1ω7c. Strain AK41(T) contained phosphatidylglycerol, phosphatidylethanolamine, an unknown aminolipid and four unknown lipids as polar lipids. Q-10 was the predominant respiratory quinone. The DNA G+C content of strain AK41(T) was 68.9 mol%. 16S rRNA gene sequence analysis indicated that strain AK41(T) was a member of the genus Rhodovulum and was closely related to Rhodovulum sulfidophilum, with 96.0 % similarity to the type strain; the 16S rRNA gene sequence similarity to the type strains of other species of the genus Rhodovulum was 93.9-95.8 %. Phylogenetic analyses indicated that strain AK41(T) clustered with the type strains of Rhodovulum marinum, Rdv. kholense, Rdv. sulfidophilum and Rdv. visakhapatnamense with sequence similarity of 95.9-96.2 %. Based on data from the current study, strain AK41(T) is proposed to represent a novel species of the genus Rhodovulum, for which the name Rhodovulum mangrovi sp. nov. is proposed. The type strain of Rhodovulum mangrovi is AK41(T) ( = MTCC 11825(T) = JCM 19220(T)).

Phaeospirillum oryzae sp. nov., a spheroplast-forming, phototrophic alphaproteobacterium from a paddy soil.

Two strains (JA317(T) and JA559) of spiral shaped, spheroplast-forming, anaerobic, gram-negative, motile purple non-sulfur bacteria were isolated from rhizosphere soils of paddy and were characterized by a polyphasic taxonomic approach. Bacteriochlorophyll a and carotenoids, rhodopin, lycopene and rhodopin glucoside, were present as photosynthetic pigments. Intracellular photosynthetic membranes were of stacked type. The major fatty acids were C(18 : 1)ω7c, C(16 : 0) and C(16 : 1)ω6c/C(16 : 1)ω7c in both strains. The genomic DNA G+C content was 63.3±0.8 mol%. The two strains were closely related (mean DNA-DNA hybridization >85 %). Phylogenetic analysis showed that the strains clustered with the species of the genus Phaeospirillum, which belongs to the family Rhodospirillaceae within the class Alphaproteobacteria. Based on 16S rRNA gene sequence analysis, strains JA317(T) and JA559 showed highest sequence similarity with the type strains of Phaeospirillum chandramohanii (98.2 %), Phaeospirillum molischianum (97.4 %) and Phaeospirillum fulvum (97.1 %) of the family Rhodospirillaceae. Strain JA317(T) can be clearly distinguished from P. chandramohanii with respect to spheroplast formation and several other morphological and physiological properties. DNA-DNA relatedness of strain JA317(T) with its closest relatives of the genus Phaeospirillum was less than 55 %. It is evident from the phenotypic, chemotaxonomic and molecular genetic evidence that strain JA317(T) represents a novel species of the genus Phaeospirillum, for which the name Phaeospirillum oryzae sp. nov., is proposed. The type strain of the species is JA317(T) ( = NBRC 104938(T)  = KCTC 5704(T)).

Marichromatium fluminis sp. nov., a slightly alkaliphilic, phototrophic gammaproteobacterium isolated from river sediment.

An anoxygenic, phototrophic gammaproteobacterium designated strain JA418(T) was isolated from a sediment sample collected from the Baitarani River, Orissa, India. The bacterium was a Gram-negative, motile rod with a single polar flagellum. Bacteriochlorophyll a and rhodopin were the major photosynthetic pigments. The organism grew best at slightly alkaline pH (8-8.5) and lacked chemotrophic growth. The major fatty acids were C(16 : 0), C(16 : 1)omega7c/C(16 : 1)omega6c and C(18 : 1)omega7c. A phylogenetic tree based on 16S rRNA gene sequence analysis showed that strain JA418(T) clusters with species of the genus Marichromatium belonging to the class Gammaproteobacteria. The highest 16S rRNA gene sequence similarities of strain JA418(T) were found with the type strains of Marichromatium gracile (95.9 %), Marichromatium indicum (95.6 %), Marichromatium purpuratum (95.5 %) and Marichromatium bheemlicum (95.6 %). The DNA base composition of strain JA418(T) was 71.4 mol% G+C (by HPLC). Based on the 16S rRNA gene sequence analysis and physiological and chemotaxonomic characteristics, strain JA418(T) is sufficiently different from other Marichromatium species to merit the description of a novel species, Marichromatium fluminis sp. nov., to accommodate it. The type strain is JA418(T) (=KCTC 5717(T) =NBRC 105221(T)).

Rhodobacter johrii sp. nov., an endospore-producing cryptic species isolated from semi-arid tropical soils.

An oval to rod-shaped, phototrophic, purple non-sulfur bacterium, strain JA192(T), was isolated from an enrichment culture of a pasteurized rhizosphere soil sample from a field cultivated with jowar (sorghum) collected from Godumakunta village near Hyderabad, India. Strain JA192(T) is Gram-negative, motile and produces endospores. Phylogenetic analysis on the basis of 16S rRNA gene sequences showed that the strain JA192(T) is closely related to Rhodobacter sphaeroides 2.4.1(T) (99.9 % sequence similarity), Rba. megalophilus JA194(T) (99.8 %) and Rba. azotoformans KA25(T) (98.1 %) and clusters with other species of the genus Rhodobacter of the family Rhodobacteraceae. However, DNA-DNA hybridization with Rba. sphaeroides DSM 158(T), Rba. megalophilus JA194(T) and Rba. azotoformans JCM 9340(T) showed relatedness of only 38-57 % with respect to strain JA192(T). On the basis of 16S rRNA gene sequence analysis, DNA-DNA hybridization data and morphological, physiological and chemotaxonomic characters, strain JA192(T) represents a novel species of the genus Rhodobacter, for which the name Rhodobacter johrii sp. nov. is proposed. The type strain is JA192(T) (=DSM 18678(T) =JCM 14543(T) =MTCC 8172(T)).

Roseomonas aestuarii sp. nov., a bacteriochlorophyll-a containing alphaproteobacterium isolated from an estuarine habitat of India.

Two strains (JC17(T) and JC19a) of orange pigmented bacteria were isolated from an estuarine sample. Cells of both the strains were Gram-negative coccobacilli, non-motile, non-spore forming and strictly aerobic. Chemo-organoheterotrophy was the growth mode for both strains and was possible on a wide range of organic compounds. Strains were non-hemolytic and contained low levels of BChl-a and carotenoids. The fatty acids (>1.0%) comprised C(18:1)omega7c, C(16:1)omega7c/iso-C(15:0)2OH, C(16:0), C(16:0) 3-OH, C(18:1)2OH, C(16:1)omega5c, and C(19:0) cycloomega8c. The genomic DNA G+C content of strain JC17(T) was 66.2mol%. A phylogenetic tree based on 16S rRNA gene sequence analysis showed that strains JC17(T) and JC19a had the highest similarity to members of the genus Roseomonas and were closely related to Roseomonas cervicalis CIP104027(T) (96.4%) and Roseomonas ludipueritiae CIP107418(T) (96.3%) of the family Acetobacteraceae within the class Alphaproteobacteria. Strains JC17(T) and JC19a shared 100% 16S rRNA gene sequence similarity, were phenotypically (morphological, physiological, biochemical characters) identical and had closely related genomes (85% DDH). Based on polyphasic taxonomic data, strain JC17(T) is classified as a novel species of the genus Roseomonas for which the name Roseomonas aestuarii sp. nov. is proposed. The type strain is JC17(T) (=CCUG 57456(T) =KCTC 22692(T) =NBRC105654(T)).

Phaeospirillum chandramohanii sp. nov., a phototrophic alphaproteobacterium with carotenoid glycosides.

A Gram-negative, spiral-shaped, phototrophic, purple non-sulfur bacterial strain, designated JA145T, was isolated from a freshwater habitat. Cells of strain JA145T were motile by means of a monopolar flagellum. Intracellular photosynthetic membranes were of the stacked type. Bacteriochlorophyll a and the carotenoid lycopene and its glucosides were present as photosynthetic pigments. There was no vitamin requirement for strain JA145T. The predominant cellular fatty acids were C16:1omega7c/C16:1omega6c (22.24%), C16:0 (22.97%) and C18:1omega7c (43.24%). Phylogenetic analysis based on 16S rRNA gene sequences showed that strain JA145T clustered with species of the genus Phaeospirillum, in the class Alphaproteobacteria. The highest sequence similarities of strain JA145T were found with the type strains of Phaeospirillum fulvum (96.12%) and Phaeospirillum molischianum (96.19%). Based on the 16S rRNA gene sequence analysis and the morphological and physiological characteristics, strain JA145T is considered to represent a novel species, for which the name Phaeospirillum chandramohanii sp. nov. is proposed. The type strain is JA145T (=JCM 14933T=KCTC 5703T=NBRC 104961T).

Carotenoids and carotenogenesis in cyanobacteria: unique ketocarotenoids and carotenoid glycosides.

Cyanobacteria grow by photosynthesis, and necessarily contain chlorophyll and carotenoids, whose main functions are light harvesting and photoprotection. In this review, we discuss the carotenoids, carotenogenesis pathways, and characteristics of carotenogenesis enzymes and genes in some cyanobacteria, whose carotenogenesis enzymes have been functionally confirmed. In these cyanobacteria, various carotenoids have been identified, including the unique ketocarotenoids, echinenone and 4-ketomyxol; and the carotenoid glycosides, myxol glycosides and oscillol diglycosides. From these findings, certain carotenogenesis pathways can be proposed. The different compositions of carotenoids among these species might be due to the presence or absence of certain gene(s), or to different enzyme characteristics. For instance, two distinct beta-carotene ketolases, CrtO and CrtW, are properly used in two pathways depending on the species. One beta-carotene hydroxylase, CrtR, has been identified, and its substrate specificities vary across species. At present, functionally confirmed genes have been found in only a few species, and further studies are needed.

Phytoene desaturase, CrtI, of the purple photosynthetic bacterium, Rubrivivax gelatinosus, produces both neurosporene and lycopene.

Biosynthetic pathways for carotenoids in the purple photosynthetic bacterium, Rubrivivax gelatinosus, which synthesizes spirilloxanthin in addition to spheroidene and OH-spheroidene, were investigated by means of genetic manipulation. A phytoene desaturase gene (crtI) found in the photosynthesis gene cluster of this bacterium was expressed in an Escherichia coli strain that can produce phytoene. Both neurosporene and lycopene were synthesized in the recombinant, probably by three- and four-step desaturation reactions of CrtI. A mutant of RVI: gelatinosus lacking the crtI gene produced only phytoene, indicating that this organism had no other phytoene desaturases. When the crtI deletion mutant was complemented by the three-step phytoene desaturase of Rhodobacter capsulatus, spirilloxanthin and its precursors were not synthesized, although spheroidene and OH-spheroidene were accumulated. It was concluded that neurosporene and lycopene are produced by a single phytoene desaturase in RVI: gelatinosus resulting in the synthesis of spheroidene and spirilloxanthin, and that there are no pathways for spirilloxanthin synthesis via spheroidene.

Photophysical characterization of natural cis-carotenoids.

By means of steady-state fluorescence spectroscopy we explore the photophysics of two lowest lying singlet excited states in two natural 15-cis-carotenoids, namely phytoene and phytofluene, possessing three and five conjugated double bonds (N), respectively. The results are interpreted in relation to the photophysics of all-transcarotenoids with varying N. The fluorescence of phytofluene is more Stokes-shifted relative to that of phytoene, and is ascribed to the forbidden S1-->S0 transition, with its first excited singlet state (S1) lying 3340 cm-1 below the dipole allowed second excited singlet state (S2), at 77 K. For phytoene the S2 and S1 potential surfaces are closer in energy, probably giving rise to the mixed S2 and S1 fluorescence characteristics. The origin of phytoene fluorescence is discussed and is suggested to be due to the S1-->S0 transition; with the S1 state located 1100 cm-1 below S2 at 77 K. The dependence of the fluorescence quantum yield on temperature and viscosity shows that large amplitude molecular motions are involved in the radiationless relaxation process of phytoene. The transition dipole moment of absorption and emission are parallel in phytoene and nonparallel in phytofluene.

Myxoxanthophyll in Synechocystis sp. PCC 6803 is myxol 2'-dimethyl-fucoside, (3R,2'S)-myxol 2'-(2,4-di-O-methyl-alpha-L-fucoside), not rhamnoside.

We identified the molecular structures of the carotenoids in Synechocystis sp. PCC 6803. Myxoxanthophyll in this cyanobacterium was myxol 2'-dimethyl-fucoside, (3R,2'S)-myxol 2'-(2,4-di-O-methyl-alpha-L-fucoside). The sugar moiety of the pigment was not rhamnose but dimethylated fucose, which has not been reported in carotenoid glycosides. The other carotenoids were beta-carotene, (3R,3'R)-zeaxanthin, echinenone, (3'R)-3'-hydroxyechinenone and deoxymyxol 2'-dimethyl-fucoside, (2'S)-deoxymyxol 2'-(2,4-di-O-methyl-alpha-L-fucoside). Generally, the group of polar carotenoids in cyanobacteria is referred to as myxoxanthophyll, and the structure is considered to be myxol 2'-rhamnoside. Since the name myxoxanthophyll can not specify the sugar moiety and the identification of the sugar moiety is unfeasible in many cyanobacteria, we propose the following naming convention: when the sugar moiety is unknown, the name is myxol glycoside, when known, as in the case of rhamnose and alpha-L-fucose, they should be named myxol 2'-rhamnoside and myxol 2'-alpha-L-fucoside, respectively.

Detailed biosynthetic pathway to decaprenoxanthin diglucoside in Corynebacterium glutamicum and identification of novel intermediates.

Carotenogenic mutants of Corynebacterium glutamicum were analyzed for their carotenoid content. Mutant MV10 accumulated the same carotenoids as the wild-type, decaprenoxanthin, decaprenoxanthin monoglucoside, and (2R,6R,2'R,6'R)-decaprenoxanthin di-(beta-D)-glucoside, but in three-fold higher amounts. In addition, decaprenoxanthin diglucoside fatty acid esters and the intermediates nonaprene, 2-(3-methyl-2-butenyl)-epsilon,psi-carotene, and sarcinene, 2,2'-bis(3-methyl-2-butenyl)-epsilon,epsilon-carotene were identified as minor carotenoids. The pink mutants MV40 and MV60 synthesized only lycopene. From another pink mutant, MV70, novel C(50)-carotenoids were isolated. By NMR and mass spectroscopy, nonaflavuxanthin, 2-(4-hydroxy-3-methyl-2-butenyl)-1,16-didehydro-1,2-dihydro-psi,psi-carotene, and flavuxanthin, 2,2'-bis(4-hydroxy-3-methyl-2-butenyl)-1,16,1',16'-tetradehydro-1,2,1',2'-tetrahydro-psi,psi-carotene, were identified. The identification of these intermediates revealed the detailed pathway for the formation of decaprenoxanthin derivatives in Corynebacterium glutamicum.

Dihydroxylycopene diglucoside diesters: a novel class of carotenoids from the phototrophic purple sulfur bacteria Halorhodospira abdelmalekii and Halorhodospira halochloris.

The carotenoids in Halorhodospira abdelmalekii and Halorhodospira halochloris were analyzed by spectroscopic methods. The carotenoid composition of the two species was almost the same. Both species contained substantial amounts of unusual carotenoid glycoside fatty acid esters, which have been found for the first time in phototrophic purple bacteria. Methoxy-hydroxylycopene glucoside was a major component, and dihydroxylycopene diglucoside and dihydroxylycopene diglucoside diester were also found. Lycopene, rhodopin, and 3,4,3',4'-tetrahydrospirilloxanthin were present in very small amounts. Methoxy, glucosyl, and glucosyl ester groups were observed as substituents at the positions of the two original hydroxyl groups of dihydroxylycopene and made up approximately 20, 50, and 20%, respectively, of the total end groups (100%). The fatty acid components of the three carotenoid glucoside esters were the same (C12:0 and C14:1) and were rare in the cellular lipids of the two species.

Absence of carotenes and presence of a tertiary methoxy group in a carotenoid from a thermophilic filamentous photosynthetic bacterium Roseiflexus castenholzii.

We identified pigments in a thermophilic filamentous photosynthetic bacterium Roseiflexus castenholzii strain HL08. We detected neither bacteriochlorophyll (BChl) c nor carotenes in this bacterium cultured under the aerobic dark and the anaerobic light conditions, which may correspond to its lack of chlorosomes. In the cells cultured under the aerobic dark conditions, the carotenoids were derivatives of keto-gamma-carotene, and the major ones were methoxy-keto-myxocoxanthin and keto-myxocoxanthin glucoside fatty acid ester. Although the tertiary methoxy group at C-1' and the double bond at C-3',4' in the psi end group of carotenoid, such as spirilloxanthin, have only been found in purple bacteria, this was the first such report in other bacterial groups. The fatty acid moiety was composed of iso fatty acids, which were rare in the cellular lipids. In the cells cultured under the anaerobic light conditions, in addition to these keto-carotenoids, we also found non-oxidized carotenoids (derivatives of gamma-carotene). Concerning the esterifying alcohol of BChl a, we found a substantial amount of geranylgeraniol, although the major component was phytol. The existence of these pigments makes this bacterium unique among the known species in CHLOROFLEXACEAE.

Identification of a gene required for cis-to-trans carotene isomerization in carotenogenesis of the cyanobacterium Synechocystis sp. PCC 6803.

A disruptive mutant of the sll0033 gene of the cyanobacterium Synechocystis sp. PCC 6803 produced primarily cis carotenes and small amounts of all-trans carotenes, but no xanthophylls, under dark conditions. Under light conditions, however, it produced normal carotenoids, that were the same as those produced by wild-type cells grown under both light and dark conditions. When the mutant cells cultured under dark conditions were irradiated, cis-isomers of carotenes were converted to all-trans lycopene. These findings demonstrate that this gene, designated crtH, is involved in the isomerization of cis-carotenes to all-trans forms in dark conditions, and that cis-carotenes were also converted to all-trans forms under light conditions by photoisomerization.

Accumulation of unusual carotenoids in the spheroidene pathway, demethylspheroidene and demethylspheroidenone, in an alkaliphilic purple nonsulfur bacterium Rhodobaca bogoriensis.

Carotenoids extracted from cells of a novel alkaliphilic purple nonsulfur bacterium Rhodobaca bogoriensis strain LBB1 included unusual carotenoids in the spheroidene pathway; demethylspheroidene, demethylspheroidenone, neurosporene and spheroidenone. Spheroidene was present in only small amounts, and the demethyl-carotenoids demethylspheroidene and demethylspheroidenone predominated in phototrophic cultures. Furthermore, the keto-carotenoids spheroidenone and demethylspheroidenone constituted nearly half of the total carotenoids, even in strict anaerobic phototrophic cultures. Spheroidenone was, however, the sole carotenoid in aerobic cultures. Phototrophic cultures of Rbc. bogoriensis were yellow in colour and quite distinct from the brown-red colour of cultures of Rhodobacter species. The carotenogenesis pathways of Rhodobaca and Rhodobacter species are compared with special reference to two key enzymes of the spheroidene pathway, CrtA and CrtF, whose activities are thought to be responsible for the unusual carotenoid composition of Rhodobaca. This bacterium also contained bacteriochlorophyll a (p) and ubiquinone-10.

Novel hydroxycarotenoids with improved antioxidative properties produced by gene combination in Escherichia coli.

We have used combinatorial biosynthesis to synthesize novel lipophilic carotenoids that are powerful cellular antioxidants. By co-expressing three different carotenoid desaturases in combination with a carotenoid hydratase, a cyclase, and a hydroxylase on compatible plasmids in Escherichia coli, we synthesized four novel carotenoids not previously detected in biological material or chemically synthesized. Their identification was based on their relative retention times on HPLC, spectroscopic properties, molecular weights, number of hydroxy groups, and 1H-NMR spectra. The carotenoids were designated as 1-HO-3', 4'-didehydrolycopene, 3, 1'-(HO)2-gamma-carotene, 1,1'-(HO)2-3, 4, 3', 4'-tetradehydrolycopene, and 1, 1'-(HO)2-3, 4-didehydrolycopene. These novel acyclic derivatives differ from structurally related compounds by extension of the conjugated polyene chain as well as additional hydroxy groups at position C-1'. We determined their antioxidative activity in a liposome-membrane model system, which showed that their ability to protect against photooxidation and radical-mediated peroxidation reactions was linked to the length of the conjugated double-bond system and the presence of a single hydroxy group. The protection of membrane degradation was superior to the related 1-HO and 1, 1'-(HO)2 lycopene derivatives, making them interesting pharmaceutical candidates.

Characterization of carotenes in a combination of a C(18) HPLC column with isocratic elution and absorption spectra with a photodiode-array detector.

Carotenes have attracted much attention in recent years for their biological function in processes such as photosynthesis. The characterization of carotenes is difficult, however, because they consist of only carbon and hydrogen atoms, without oxygen. In the present study, we systematically examined the chemical structures of more than 30 carotenes, including most of the carotenes found in phototrophic organisms, and observed their elution order using a Novapak C(18) HPLC column with simple isocratic elution. The elution order of the carotenes was C(30), C(40),C(45) then C(50). The C(40) carotenes with fewer conjugated double bonds (N) had longer retention times. With respect to the end groups, the carotenes eluted in the following order: phi, Psi, in then beta end groups. Furthermore, absorption spectra in the HPLC eluent used were recorded with a photodiode-array detector. A greater N value was associated with a longer absorption maximum wavelength. Since the conjugated end groups (phi and beta) influenced the absorption spectra and the non-conjugated end groups (Psi and in) did not, the number of conjugated end groups (zero, one and two) was clearly distinguishable. Therefore, the chemical structures of carotenes can be easily determined by a combination of the HPLC retention times and the absorption spectra.

Papain-hydrolyzed pork meat reduces serum cholesterol level and premature atherosclerosis in dietary-induced hypercholesterolemic rabbits.

The effects of the low-molecular-weight fraction of papain-hydrolyzed pork meat (LMF) on the plasma cholesterol level and the generation of atherosclerosis were studied in rabbits fed a cholesterol-enriched diet. In LMF-fed rabbits, the plasma and liver cholesterol concentrations were both significantly lower (p<0.0 1) than in rabbits fed untreated pork meat (PM). Similarly, the cholesterol concentrations of the chylomicron and VLDL fractions were significantly lower in LMF-fed rabbits than in rabbits fed PM. Deposition of lipid in transverse sections of the aortic arch was significantly less in rabbits fed LMF than in those fed PM. Electron microscopic studies revealed preventive effects against premature atherosclerotic lesions in the aorta of rabbits fed LME These results indicate that LMF has a hypocholesterolemic action and preventive effects against premature atherosclerosis.