Effects of desmethylimipramine and diphenylpropene derivatives on synthesis of rat brain lipids in vivo

The effects of antidepressant compounds on the synthesis of brain lipids from [1-¹⁴C] acetate $\text{in vivo}$ in 15 day old rats have been investigated. Compounds used included the drug desmethylimipramine (DMI), the tetrabenazine antagonist 3-methylamino-1:1-diphenylprop-1-ene (II) and the primary (I) and tertiary (III) amine analogues of (II). Compound (II), the most potent tetrabenazine antagonist in the diphenylpropene series, significantly increased lipogenesis, whereas the remaining compounds did not. The results from fractionation of the lipid extract from rats treated with (II) indicated that the incorporation of radioactivity from [1-¹⁴C] acetate increased proportionally in all neutral lipids and phospholipids. Tetrabenazine also increased brain lipogenesis $\text{in vivo}$ and altered the distribution within lipid classes of radioactivity from [1-¹⁴C] acetate. Using [¹⁴C] labelled compound, the concentration of (II) in the brain under the present experimental conditions has been determined.     

Ozonolytic cleavage of authentic and pancreatic dolichyl mannopyranosyl phosphates: determination of sugar configuration in the fragments with alpha- and beta-mannosidases.

Exposure of authentic dolichyl α-D-[¹⁴C]mannopyranosyl phosphate ($\text{I}$) or calf pancreas dolichyl [¹⁴C]mannopyranosyl phosphate ($\text{II}$) to ozone at ?70° in pentane followed by treatment with triphenylphosphine gave water-soluble fragments in 65–95% yield. The radioactive products obtained were similar; the major fragment had a mobility on tlc greater than that of mannose but lower than that of citronellyl β-D-mannopyranosyl phosphate. The electrophoretic behavior of the fragments indicated that they possessed intact phosphodiester linkages. α-Mannosidase released [¹⁴C]mannose from the fragments of $\text{I}$ but not from the fragments of $\text{II}$; however, the latter were susceptible to β-mannosidase indicating that the pancreatic mannolipid contains a β-linked mannosyl residue.     

Amphomycin inhibits the incorporation of mannose and GlcNAc into lipid-linked saccharides by aorta extracts

Amphomycin inhibits the incorporation of mannose from GDP-[¹⁴C]mannose and GlcNac from UDP-[³H]GlcNAc into lipid-linked saccharides by either a particulate or a solubilized enzyme fraction from pig aorta. The solubilized enzyme was much more sensitive to the antibiotic than was the particulate fraction with 50% inhibition being observed at 8–15 μg of amphomycin. Although the antibiotic inhibited mannose transfer from GDP-[¹⁴C]mannose into mannosyl-phosphoryl-dolichol, lipid-linked oligosaccharides and glycoprotein, the synthesis of mannosyl-phosphoryl-dolichol was much more sensitive to amphomycin. Amphomycin also inhibited the incorporation of mannose from GDP-[¹⁴C]mannose into mannosyl-phosphoryldecaprenol in particulate extracts of $\text{Mycobacterium smegmatis}$.     

Isolation of 4α-methyl-5α-Ergosta-8, 24 (28)-dien-3β-ol from ester fractions of nutritionally deprived neurospora crassa

A method of isolating pure fractions of 4α-methyl-5α-ergosta-8, 24 (28)-dien-3β-ol for sterol intermediate studies is described. Starvation cultures of $\text{Neurospora crassa}$ readily incorporate exogenous mevalonic acid into the sterol ester fraction. Isolation involves a simple solvent extraction and two chromatograms. Only the ester fraction yielded the required purity. Radioactive 4α-methyl-5α-ergosta-8, 24 (28)-dien-3β-ol is readily produced from DL-[2-¹⁴C] mevalonic acid.     

THE BIOSYNTHESIS OF CHOLESTEROL AND OTHER STEROLS BY BRAIN TISSUE: DISTRIBUTION IN SUBCELLULAR FRACTIONS AS A FUNCTION OF TIME AFTER INJECTION OF [2-14C]MEVALONIC ACID, SODIUM [2-14C]ACETATE AND [U-14C]GLUCOSE INTO 15-DAY-OLD RATS

The distribution of [¹⁴C]-labelled material into subcellular fractions of 15-day-old rat brain was studied at 2 and 24 h following intraperitoneal and intracerebral injection of [2-¹⁴C]sodium acetate, [U-¹⁴C]glucose and [2-¹⁴C]mevalonic acid respectively. The total quantity of labelled isoprenoids in the brain was, except for glucose, greater when the precursor was administered intracerebrally. The intraperitoneal route was more advantageous in the case of [U-¹⁴C]glucose. The subcellular distribution of both labelled total isoprenoid material and sterol was distinct for each labelled precursor. Intracerebrally injected [U-¹⁴C]glucose at both time periods studied suggested no dominance of labelling in any fraction. After intraperitoneal injection of [U-¹⁴C]glucose the microsomes were more prominently labelled. Both methods of administration of sodium [2-¹⁴C]acetate resulted in heavy labelling of the myelin fraction after 24 h. The total labelled isoprenoids resided mainly in the microsomes 24 h after injection of [2-¹⁴C]mevalonic acid. Labelled sterol was found to be localized more in the myelin and microsomal fractions for all three precursors than was the labelled total isoprenoids. Depending on the type of experiment to be conducted, each of these precursors can give different results, which must be interpreted accordingly.     

Isolation and partial characterization of a mucin-type glycoprotein from plasma membranes of human melanoma cells

Plasma membranes were isolated from HM7 melanoma cells grown in the presence of [³H]glucosamine and Na₂³⁵SO₄ or [³H]mannose and [¹⁴C]glucosamine. The labelled glucoconjugates were solubilized with 0.6 M lithium diiodosalicylate/0.5% Triton X-100. Fractionation of glycoconjugates by repeated chromatography on columns of Sepharose CL-6B and DEAE-Sepharose and by affinity chromatography on WGA-Sepharose yielded three radiochemically homogenous glycoproteins. One of these having an apparent molecular weight of 100 000 was found to contain clusters of (AcNeu)_{1 or in2} å [Gal å GalNAc] linked $\text{O-}\text{glycosidically}$ to the protein. One other glycoprotein contained both $\text{O-}\text{glycosidically}$ and $\text{N-}\text{glycosidically-linked}$ oligosaccharides, and the third contained only $\text{N-}\text{glycosidically-linked}$ carbohydrates. Preliminary results indicate that the 100 000 molecular weight mucin-type glycoprotein is present in significantly reduced quantities in cultured human fetal uveal melanocytes. Further, the bulk of the glycoproteins from the melanocytes were of lower molecular size compared to those from the melanoma cells.     

The biosynthesis of lubimin from [1-14C]isopentenyl pyrophosphate by cell-free extracts of potato tuber tissue inoculated with an elicitor preparation from Phytophthora infestans

A cell-free enzyme system, which catalyses the incorporation of radiolabel from [$\text{1}\text{2}$-¹⁴C]isopentenyl pyrophosphate into the sesquiterpenoid phytoalexin lubimin, has been prepared from tuber tissue of Solanum tuberosum inoculated with an elicitor preparation from Phytophthora infestans. Biosynthesis of lubimin is optimum at pH 7.$\text{3}\text{2}$-7.5 and is dependent upon Mg²⁺ and NADPH. Lubimin labelling by cell-free enzyme system prepared from tissue 48 hr after treatment with elicitor rises rapidly to a maximum over the first 30 min of incubation and does not decline for a further 150 min. The biosynthetic capacity for lubimin in cell free extracts can be observed as early as 3 hr after inoculation of tuber tissue, and rises to a maximum at about 48 hr after treatment, declining thereafter. Lubimin labelling is inhibited by iodoacetamide, the effect of which is reversed by 3,3-dimethylallylpyrophosphate. Preliminary observations on the cell-free system show that it will also catalyse the incorporation of [2-¹⁴C]mevalonic acid into lubimin in the presence ofan ATP-generating system.     

Dietary effects on the formation of dolichyl monophosphate mannose by microsomal preparations of rat adipose tissue

Microsomal preparations from rat adipose tissue catalyse the transfer of [¹⁴C]mannose from GDP-[¹⁴C]mannose to an endogenous acceptor forming a [¹⁴C]mannosyl lipid. The mannosyl lipid co-chromatographs with hen oviduct dolichyl monophosphate β-mannose on three solvent systems. It is stable to mild alkaline hydrolysis, but strong alkaline treatment yields a compound that co-migrates with mannose 1-phosphate. The mannosyl lipid is labile to mild acid hydrolysis, yielding [¹⁴C]mannose. Formation of the compound is reversible by GDP, but not GMP, and is stimulated by exogenous dolichyl phosphate.

The kinetics of transfer of [¹⁴C]mannose from GDP-[¹⁴C]mannose to form dolichyl monophosphate mannose were studied by using preparations derived from rats fed on one of four diets: G (high glucose), L (high lard), F (fructose) or GC (high glucose, 0.9% cholesterol). The K_m and V_max. values for transfer from GDP-mannose were virtually indistinguishable in the four preparations.

In the absence of exogenous dolichyl phosphate, the largest amount of transfer of [¹⁴C]mannose into the mannosyl lipid was observed with preparations from fructose-fed animals. Preparations from glucose-fed animals showed about 60% as much transfer, whereas membranes from rats fed the other diets showed intermediate values between the fructose- and glucose-fed animals. The inclusion of cholesterol in the glucose diet elicited an increase in transfer of mannose.

Under conditions of saturating exogenous dolichyl phosphate, preparations from lard-fed animals have 1.5 times as much enzyme activity as do preparations from animals fed the other three diets.

     

In vivo biosynthesis of -linolenic acid in plants

[1-¹⁴C]acetate was readily incorporated into unsaturated fatty acids by leaf slices of spinach, barley and whole cells of $\text{Chlorella}\text{pyrenoidosa}$ and $\text{Candida}\text{bogoriensis}$. In these systems the [¹⁴C] label in newly synthesized oleate and linoleate was approximately equally distributed in the C_1–9 and the C_10–18 fragments obtained by reductive ozonolysis of these acids, whereas in a-linolenic acid over 90% of the total [¹⁴C] was localized in the C_1–9 fragment. While [1-¹⁴C]oleic acid was converted by whole cells of $\text{Chlorella}$ to [1-¹⁴C]linoleic and [1-¹⁴C]linolenic acids, [U-¹⁴C]oleic acid yielded [U-¹⁴C]linoleic acid but a-linolenic acid was labeled only in the carboxyl terminal carbon atoms. When spinach leaf slices were supplied with carboxyl labeled octanoic, decanoic, dodecanoic, tetradecanoic and octadecanoic acids, only the first three acids were converted to a-linolenic acids while the last two acids were ineffective. Thus we suggest that (a) linoleic acid is not the precursor of a-linolenic acid and (b) 12:3(3, 6, 9) is the earliest permissible trienoic acid which is then elongated to a-linolenic acid.     

The involvement of sucrose,glucose and other metabolites in the synthesis of triterpenes and dopa in the laticifers of Euphorbia lathyris

A quantitative triterpene analysis was made of latex stem tissue of Euphorbia lathyris. Young plants seedlings of E. lathyris were incubated with various labelled precursors. Incorporation into triterpenes was obtained from [2-¹⁴C]mevalonic acid, [1-¹⁴C]acetate, [3-¹⁴C]pyruvate, [U-¹⁴C]sucrose, [U-¹⁴C]glucose, [U-¹⁴C]xylose, [U-¹⁴C]glyoxylate, [2,3-¹⁴C]succinic acid, [1-¹⁴C]glycerol [U-¹⁴C]serine. Both sugars tyrosine appeared to be effective precursors in DOPA synthesis inside the laticifers. Exogenously supplied mevalonic acid was only involved in triterpene synthesis outside the laticifers. GC-RC of triterpenes synthesized from [U-¹⁴C]glucose revealed the origin of these compounds in the latex. The labelled triterpenes obtained after incorporation of the other mentioned labelled precursors were only partly synthesized in the laticifers. For quantitative data on latex triterpene synthesis seedlings were incubated with [U-¹⁴C]sucrose, [U-¹⁴C]glucose, [U-¹⁴C]xylose [1-¹⁴C]acetate in the presence of increasing amounts of unlabelled substrate. From the amount of ¹⁴C incorporated into the triterpenes the amount of substrate directly involved in triterpene synthesis was calculated, as was the absolute triterpene yield. Sucrose showed the highest triterpene yield, equivalent to the daily increase of the triterpene content of growing seedlings. The possible significance of the other precursors in triterpene synthesis in the laticifers is discussed.     

RATE OF STEROL FORMATION BY RAT BRAIN GLIA AND NEURONS IN VITRO AND IN VIVO

The ability of 11-day-old rat glial and neuronal cells to biosynthesize sterol was studied as a function of time in vivo and in vitro. The in vitro experiments utilized [2-¹⁴C]mevalonic acid as precursor. Glial-enriched cell preparations demonstrated a greater ability to incorporate [2-¹⁴C]mevalonic acid into isoprenoid material than did neuronal-enriched preparations. Approximately 4 h were required for maximal uptake of labelled mevalonate by the glial preparations. Further metabolism of the isoprenoid material, involving squalene turnover and sterol demethylation, was still evident even after 15 h of incubation. In vivo, sterol biosynthesis was studied by intraperitoneal injection of sodium [2-¹⁴C]acetate and [U-¹⁴C]glucose, sacrifice of the animals at 2 or 24 h, subsequent isolation of glial- and neuronal-cell enriched fractions and analysis of labelled isoprenoid material. Glial-enriched fractions again contained the bulk of the labelled isoprenoid material.     

Biosynthesis of 3-methylpentacosane in the cockroach Periplaneta americana.

The biosynthesis of 3-methylalkanes was investigated in the cockroach $\text{Periplaneta americana}$. Between 0.2 and 0.3 percent of the labelled acetate and propionate injected into the insect was incorporated into the cuticular hydrocarbons, compared to 0.01 percent for labelled isoleucine. Twenty-three ± four percent of the [2-¹⁴C]acetate, 42 ± 3 and 44 ± 4 percent of the [2-¹⁴C] and [3-¹⁴C]propionate, and 75 ± 5 percent of the [1-¹⁴C]propionate incorporated into the cuticular hydrocarbons was found in 3-methylpentacosane. These results indicate that propionate serves as the source of the branching methyl group, suggesting a pathway in which this precursor is incorporated during the penultimate step in 3-methylalkane biosynthesis in insects.     

Amino acid uptake by yeasts: IV. Effect of thiol reagents on l-leucine transport in Saccharomyces cerevisiae

(1) $\text{N-}\text{Ethylmaleimide}$ (a penetrating SH- reagent) inactivated l-[¹⁴C]leucine entrance (binding and translocation) into Saccharomyces cerevisiae, the extent of inhibition depending on the time of preincubation with $\text{N-}\text{ethylmaleimide}$, $\text{N-}\text{ethylmaleimide}$ concentration, the amino acid external and internal concentration, and the energization state of the yeast cells. With d-glucose-energized yeast, $\text{N-}\text{ethylmaleimide}$ inhibited l-[¹⁴C]leucine entrance in all the assayed experimental conditions, but with starved yeast and low (0.1 mM) amino acid concentration, it did not inhibit l-[¹⁴C]leucine binding, except when the cells were preincubated with l-leucine. With the rho^? respiratory-deficient mutant (energized cells), $\text{N-}\text{ethylmaleimide}$ inhibited l[¹⁴C]leucine entrance as with the energized wild-type, though to a lesser extent. (2) Analysis of the $\text{N-}\text{ethylmaleimide}$ effect as a function of l-[¹⁴C]leucine concentration showed a significant decrease of $\text{J}{}_{\mathrm{<mtext>max</mtext>}}$ values of the high- (S₁) and low- (S₂) affinity amino acid transport systems, but $\text{K}{}_{\mathrm{<mtext>T</mtext>}}$ values were not significantly modified. (3) When assayed in the presence of d-glucose, $\text{N-}\text{ethylmaleimide}$ inhibition of d-glucose uptake and respiration contributed significantly to inactivation of l-[¹⁴C]leucine entrance. Pretreatment of yeast cells with 2,4-dinitrophenol enhanced the effect of l-[¹⁴C]leucine binding and translocation. (4) Bromoacetylsulfanilic acid and bromoacetylaminoisophthalic acid, two non-penetrating SH- reagents, did not inactivate l-[¹⁴C]leucine entrance, while $\text{p-}\text{chloromercuribenzoate}$, a slowly penetrating SH- reagent, inactivated it to a limited extent. When compared with the effect of $\text{N-}\text{ethylmaleimide}$, these negative results indicate that thiol groups of the l-[¹⁴C]leucine carrier were not exposed on the outer surface of the yeast cell permeability barrier.     

A succinylated mannan in the membrane system of Micrococcus lysodeikticus

A mannan has been isolated from membranes of $\text{Micrococcus lysodeikticus}$ (NCTC 2665) grown in the presence of [1, 4 ¹⁴C] succinic acid. Hydrolysis of this polysaccharide with 0.1 N NaOH yielded a compound soluble in both diethyl ether and distilled water. This component was identified as [¹⁴C] succinic acid by paper chromatography of both the free acid and of its corresponding hydroxamic acid derivative. Semiquantitative analysis indicated an ester-linked succinic acid content for mannan of approximately 2.5%.     

Synthesis and glycosylation of the MOPC-46B immunoglobulin in kappa chain in Xenopus laevis oocytes.

Polyadenylated mRNA isolated from MOPC-46B plasmacytoma, which secretes a glycosylated kappa chain, was injected into $\text{Xenopus laevis}$ oocytes. Analysis of the resulting product showed that [1-¹⁴C]mannose was incorporated into the MOPC-46B kappa chain. Light chains synthesized in oocytes injected with mRNA from MOPC-321 plasmacytoma, which secretes a nonglycosylated kappa chain, failed to incorporate label from [1-¹⁴C]mannose. Thus, protein glycosylation in the oocyte is apparently specific in that carbohydrate is incorporated only into the kappa chain synthesized as a glycoprotein by myeloma cells. It is thus evident that the general signals for glycosylation have remained stable during independent evolution of the amphibia and mammalia.     

Biosynthesis of -linolenic acid by disrupted spinach chloroplasts

A disrupted spinach chloroplast preparation readily synthesized $\text{[}{}^{14}\text{C]α-linolenate}$ from [2-¹⁴C]acetate under anaerobic conditions. It can be shown by degradation data that [¹⁴C]oleate is not a precursor of [¹⁴C]linolenate and that $\text{cis}$ 7,10,13-hexadecatrienoic acid is the probable immediate precursor of the [¹⁴C]linolenate.     

Cell-free labeling in thyroid rough microsomes of lipid-linked and protein-linked oligosaccharides: I. Mannosylated units

In order to evaluate the possibility in a pig thyroid rough microsomal system of a transfer of pre-assembled sugar cores from sugar-lipids to protein, we have examined after incubation with GDP-[¹⁴C]Man the compounds bearing labeled saccharides and have determined some properties of their released saccharide moieties. The [¹⁴C]Man material specifically soluble in CHCl₃/CH₃OH/H₂O, 10 : 10 : 3, behaved on DEAE-cellulose and when treated with hot alkali and alkaline phosphatase as a lipid pyrophosphate (sometimes accompanied by some $\text{dolichol}\text{-P-[}{}^{14}\text{C}\text{]}\text{Man}$). Its saccharide moiety, released by mild acid, exhibited properties (molecular size, sensitivity to α-mannosidase, affinity for concanavalin A and charge modification introduced by a strong reductive alkaline treatment) pointing to a polymannosylated N,N′-diacetylchitobiose containing an average of nine monosaccharide units (from six to twelve). The [¹⁴C]mannosylated glycoproteins have represented all the polymeric label remaining after lipid extraction. From the susceptibility of their pronase glycopeptides to a differential reductive alkaline hydrolysis, it was concluded that their label belonged mainly to N-glycosidically linked units. Released saccharides exhibited the same properties as those from lipids, a result substantiating the possibility raised from previous studies of a transfer of pre-assembled moieties.     

Sterol-like compound from sensory ganglia. Effect of a nerve growth factor and insulin on its biosynthesis

1. Sensory ganglia from 8-day-old chick embryos were incubated with a specific nerve growth factor and with insulin. 2. From the total lipid extract of the ganglia a compound with steroid characteristics was isolated. 3. The synthesis of this compound, measured spectrophotometrically, diminished after addition of the nerve growth factor and insulin to the incubation medium. 4. The incorporation of sodium [2-(14)C]acetate and dl-[2-(14)C]mevalonic acid into total lipids of the sensory ganglia was stimulated by the nerve growth factor and insulin, but the radioactivity of the sterol-like compound was slightly lower. The incorporation of labelled mevalonic acid either into total lipids or into the sterol-like compound was about 25% lower. 5. About 20% of the acetate incorporated into total lipids and about 87% of the mevalonic acid were recovered in the sterol-like compound.     

Biosynthesis of the prenyl side chains of plastiquinone and related compounds in maize and barley shoots

The incorporation of ¹⁴C by etiolated maize and barley shoots exposed to light of ¹⁴CO₂ and [2-¹⁴C]mevalonic acid into phylloquinone, plastoquinone, ubiquinone, α-tocopherolquinone and α-tocopherol was examined. In maize (the principal tissue studied) it was demonstrated that ¹⁴C from [2-¹⁴C]mevalonic acid is incorporated into phylloquinone, plastoquinone and ubiquinone. α-Tocopherol and α-tocopherolquinone, although undoubtedly labelled from this substrate, were not purified completely. As expected, ¹⁴C from ¹⁴CO₂ was incorporated into all components examined. Ozonolytic degradation studies showed that ¹⁴C from [2-¹⁴C]mevalonic acid was incorporated specifically into the prenyl side chains of plastoquinone and ubiquinone, and from this it was inferred that mevalonic acid can be regarded as the specific distal precursor to the prenyl portions of all terpenoid quinones occurring in plant tissues. From a comparison of the relative incorporation of ¹⁴C from ¹⁴CO₂ and [2-¹⁴C]mevalonic acid into the intra- and extra-chloroplastidic terpenoids evidence was obtained consistent with the tenet that the prenyl portions of the chloroplastidic quinones phylloquinone and plastoquinone, along with β-carotene, are biosynthesized within the confines of the chloroplast, the side chain of the extraplastidic ubiquinone and phytosterols being synthesized elsewhere within the cell. The results obtained for the incorporation of ¹⁴C from ¹⁴CO₂ and [2-¹⁴C]mevalonic acid into α-tocopherol and α-tocopherolquinone were not readily interpretable with regard to the site of synthesis of these compounds.     

The cerulenin-induced formation of 1-acyl-lysophosphatidyl glycerol in Bacillus megaterium.

When a culture of $\text{Bacillus megaterium}$ ATCC 14581, growing at 20° and treated with the fatty acid synthesis inhibitor, cerulenin, was incubated with [U-¹⁴C]palmitate, 50% of the incorporated label was found in 1-palmitoyl-lysophosphatidyl glycerol within 5 min. Most of the remaining ¹⁴C appeared in free fatty acid and phosphatidyl glycerol. By 45 min almost all of the lyso compound had disappeared and 80% of the incorporated label was found in phosphatidyl glycerol. At 20°, in the absence of cerulenin or at 35° in either its presence or absence, no labeled lysophosphatidyl glycerol could be found at any time after [U-¹⁴C]palmitate addition. The major radioactive lipid, in these cases, was always phosphatidyl glycerol. At 20°, the palmitate of phosphatidyl glycerol but not of lysophosphatidyl glycerol was readily desaturated.