In vivo conversion of 18- and 20-C essential fatty acids in rats using the multiple simultaneous stable isotope method

An important question for mammalian nutrition is the relative efficiency of C18 versus C20 essential fatty acids (EFAs) for supporting the tissue composition of n-3 and n-6 pathway end products. One specific question is whether C22 EFAs are made available to tissues more effectively by dietary alpha-linolenic acid (18:3n-3) and linoleic acid (18:2n-6) or by dietary eicosapentaenoic acid (20:5n-3) and dihomo-gamma-linolenic acid (20:3n-6). To address this question in a direct manner, four stable isotope compounds were given simultaneously in a novel paradigm. A single oral dose of a mixture of 2H5-18:3n-3, 13C-U-20:5n-3, 13C-U-18:2n-6, and 2H5-20:3n-6 was administered to rats given a defined diet. There was a preferential in vivo conversion of arachidonic acid (20:4n-6) to docosatetraenoic acid (22:4n-6) and of 22:4n-6 to n-6 docosapentaenoic acid (22:5n-6) when the substrates originated from the C18 precursors. However, when the end products docosahexaenoic acid (22:6n-3) or 22:5n-6 were expressed as the total amount in the plasma compartment divided by the dosage, this parameter was 11-fold greater for 20:5n-3 than for 18:3n-3 and 14-fold greater for 20:3n-6 than for 18:2n-6. Thus, on a per dosage basis, the total amounts of n-3 and n-6 end products accreted in plasma were considerably greater for C20 EFA precursors relative to C18.     

Effects of sterols,rat hematin and coproporphyrin on the free-living stages of Nematospiroides dubius and Nippostrongylus brasiliensis

Nematospiroides dubius, unlike the closely related nematode Nippostrongylus brasiliensis, lacks a sterol requirement for the completion of its development to the L₃ stage in an aqueous suspension of formalin-killed Escherichia coli. These larvae were justascapable of completing their development to adults in mice as those grown on charcoal-feces cultures. In addition, sterols had a toxic effect on the larvae of N. dubius which was removed by substituting a Krebs-Ringer solution for distilled water.In order to interpret the absence of a sterol requirement for the development of N. dubius larvae, an analysis of the egg lipids for both species was made. N. brasiliensis had about twice the sterol concentration of N. dubius eggs. A comparison of the development for both species showed, however, that N. brasiliensis grows more than twice as much as N. dubius to reach the L₃ larval stage. Therefore, the amount of endogenous sterol is insufficient for N. brasiliensis to develop from the egg to the L₃ stage.N. dubius and N. brasiliensis also differ in their porphyrin requirements. Unlike N. brasiliensis, coproporphyrin did not increase the size of N. dubius larvae so that they were comparable to those grown on charcoal-feces cultures. Coproporphyrin did result in larvae that were more robust in appearance than those grown without a supplement.     

Nippostrongylus brasiliensis: Increase of sialomucins reacting with anti-mucin monoclonal antibody HCM31 in rat small intestinal mucosa with primary infection and reinfection

Infections with the parasitic helminth, Nippostrongylus brasiliensis, cause changes in rat small intestinal goblet cell mucin, particularly in the peripheral sugar residues of oligosaccharide. These changes may correlate with expulsion. In this study, we examined changes in mucin oligosaccharides caused by primary infection and reinfection with N. brasiliensis, using two monoclonal antibodies, HCM31 and PGM34, that react with sialomucin and sulfomucin, respectively. Enzyme-linked immunosorbent assay of jejunal mucins showed that the relative reactivity of mucins with HCM31, but not PGM34, increased up to 16 days after primary infection and 6 days after reinfection, the times when the worms were expelled from the rats. Immunohistochemical studies confirmed that goblet cells stained with HCM31 greatly increased at the time of worm expulsion. These results indicate that the marked increase observed in HCM31-reactive sialomucins may be related to expulsion of the worms.     

The location of parasites within their hosts: The influence of surgical manipulation of the intestine and mesenteric blood supply on the dispersion of Nippostrongylus brasiliensis in the rat

In the early stages of a primary infection with N. brasiliensis in rats, adults are characteristically located about one-third of the distance anad of the pyloric sphincter. As one of a series of experimental investigations of this phenomenon, surgical manipulation of the site has been attempted. The normal site has been surgically removed, it has been reversed while keeping the blood system intact and the mesenteric arterial and venous supply has been locally restricted. In no case did surgical intervention significantly lower the number of adult parasites. In all cases there was a shift from the normal dispersion to one more orad. It has been conclusively demonstrated that there is no specific structural or physiological factor in the normal site of N. brasiliensis, which is critical for its survival. All results indicate as proposed in an earlier paper, that N. brasiliensis is a lumenal dweller and is most frequently recovered from the intestinal contents.     

The location of parasites within their hosts: The influence of host feeding and diet on the dispersion of adults of Nippostrongylus brasiliensis in the intestine of the rat

This paper is the first in a series to experimentally examine factors which determine the location of parasites within their hosts. Nippostrongylus brasiliensis is typically found one third of the way along the small intestine of the rat between days 7 and 12 of a primary infection. When rats were fasted for 2 days, N. brasiliensis became evenly distributed along the intestine and many individuals, mostly females, entered the caecum. Upon being fed again, the parasites were at their ‘normal site’ within 24 h. Experimental diets of α-cellulose, or simple dietary compounds caused the same re-distribution as did normal food. By sectioning the rat intestine when it was full of food and relating the dispersion of worms to the presence of intestinal contents, it was confirmed that N. brasiliensis is found amongst the intestinal contents of its host. The notion of a ‘site’ is critically discussed and the merit of ‘habitat’ is argued because the ‘site’ of N. brasiliensis would appear to be less a ‘place’ but rather a combination of environmental factors.     

Toxic components on the larval surface of the corn earworm (Heliothis zea) and their effects on germination and growth of Beauveria bassiana

Caprylic acid is present on the surface of corn earworm, Heliothis zea, and fall armyworm, Spodoptera fragiterda, larvae. Because caprylic acid inhibits germination of Beauveria bassiana, presence of this compound will be determined to the establishment of an infection of larvae by this fungus. Other free fatty acids present on the surface of the H. zea and S. frugiterda are tentatively identified as valeric and nonanoic acids; these also possess mycostatic activity toward B. bassiana. Depending on concentration, caprylic acid inhibits germination of conidia for different amounts of time (R. J. Smith and E. A. Grula, 1981, J. Invertebr. Pathol., 37, 222–230). We now further report that inhibition and/or growth is also related to the source of carbon, nitrogen, and energy present in the growth medium. This observation of selective toxicity in the presence of different nutrients was also observed using nonanoic acid. Our data therefore make it necessary to interpret the effects of certain fatty acids on germination and growth of B. bassiana (and probably other fungi as well) in terms of nutrients for the germination process.     

Studies of regulatory metabolism in Moniezia expansa: Glutamate,and the absence of the γ-aminobutyrate pathway

Moniezia expansa takes up radioactive glutamate from an isotonic medium, and radiocarbon appears primarily in α-ketoglutarate and succinate. Glutamate-oxaloacetate and glutamate-pyruvate transaminase activities were present in subcellular preparations; however, the enzymes of the γ-aminobutyrate pathway were absent. Further attempts to obtain indirect evidence for the operation of this pathway failed, and the metabolism of glutamate appears to take place via a preliminary transamination to α-ketoglutarate, followed by oxidation to succinate.     

Mechanism of action of Clostridium perfringens enterotoxin. Effects on membrane permeability and amino acid transport in primary cultures of adult rat hepatocytes

Purified enterotoxin from the bacterium Clostridium perfringens rapidly decreased the hormonally induced uptake of α-aminoisobutyric acid in primary cultures of adult rat hepatocytes. At 5 min after toxin addition the decrease in α-aminoisobutyric acid uptake appeared not due to increased passive permeation (estimated with l-glucose) or to increased α-aminoisobutyric acid efflux. When short uptake assay times were employed a depression of α-aminoisobutyric acid influx was observed in toxin-treated hepatocytes. The depression of α-aminoisobutyric acid influx was correlated with a rapid increase in intracellular Na⁺ (estimated using ²²Na⁺) apparently effected by membrane damage. In contrast, the uptake of cycloleucine in the presence of unlabeled α-aminoisobutyric acid (assay for Na⁺-independent amino acid uptake) by hepatocytes treated with toxin for 5 min was decreased to only a small extent or not at all depending upon experimental design. At later times, C. perfringens enterotoxin increased the exodus of l-glucose, $\text{3-O-}\text{methylglucose}$ and α-aminoisobutyric acid from pre-loaded cells indicating that the toxin effects progressive membrane damage. When enterotoxin was removed by repeated washing after 5–20 min the decay of α-aminoisobutyric acid uptake ceased and appeared to undergo recovery towards the hormonally induced control level. The degree of recovery of α-aminoisobutyric acid uptake was inverse to the length of time of exposure to toxin. Adding at 10 min specific rabbit antiserum against C. perfringens enterotoxin without medium change also reversed the effect of toxin on increased intracellular ²²Na⁺, and on the exodus (from preloaded cells) of α-aminoisobutyric acid, L-glucose, and $\text{3-O-}\text{methylglucose}$.     

Determination of isonicotinic acid in the presence of isoniazid and acetylisoniazid Studies on isonicotinic acid formation from isoniazid in isolated rat hepatocytes

In comparison with the hepatocytes obtained from intact rats and rats pretreated with phenobarbital or 3-methylchoranthrene, the amount of isonicotinic acid (INA) formed from isoniazid (INH) increased substantially after incubation at 37°C using the pretreated hepatocytes. This suggests an oxidative pathway for INA formation from INH, apart from hydrolysis. In order to explore the exact mechanism of INA formation in the hepatocytes, an HPLC assay for INA in the presence of INH and acetylisoniazid was developed. In this assay, INA was extracted after the preparation of an ion pair with tetra-n-butylammonium hydroxide, and analysed using an ODS column and a mobile phase consisting of 0.067 M potassium dihydrogenphosphate solution-methanol (96:4 v/v). The method is simple, accurate and especially suitable for INA determination after incubation of INH in isolated rat hepatocytes.     

Solution structure of native and recombinant expressed toxin CssII from the venom of the scorpion Centruroides suffusus suffusus, and their effects on Nav1.5 Sodium channels

The three-dimensional structures of the long-chain mammalian scorpion β-toxin CssII from Centruroides suffusus suffusus and of its recombinant form, HisrCssII, were determined by NMR. The neurotoxin CssII (nCssII) is a 66 amino acid long peptide with four disulfide bridges; it is the most abundant and deadly toxin from the venom of this scorpion. Both native and recombinant CssII structures were determined by nuclear magnetic resonance using a total of 828 sequential distance constraints derived from the volume integration of the cross peaks observed in 2D NOESY spectra. Both nCssII and HisrCssII structures display a mixed α/β fold stabilized by four disulfide bridges formed between pairs of cysteines: C1-C8, C2-C5, C3-C6, and C4-C7 (the numbers indicate the relative positions of the cysteine residues in the primary structure), with a distortion induced by two cis-prolines in its C-terminal part. The native CssII electrostatic surface was compared to both the recombinant one and to the Cn2 toxin, from the scorpion Centruroides noxius, which is also toxic to mammals. Structural features such N- and C-terminal differences could influence toxin specificity and affinity towards isoforms of different sub-types of Na_v channels.     

Isolation and characterization of genes from the marine microalga Pavlova salina encoding three front-end desaturases involved in docosahexaenoic acid biosynthesis

The marine microalga Pavlova salina produces lipids containing approximately 50% omega-3 long chain polyunsaturated fatty acids (LC-PUFA) such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). Three cDNA sequences, designated PsD4Des, PsD5Des, PsD8Des, were isolated from P. salina and shown to encode three front-end desaturases with Delta4, Delta5 and Delta8 specificity, respectively. Southern analysis indicated that the P. salina genome contained single copies of all three front-end fatty acid desaturase genes. When grown at three different temperatures, analysis of fatty acid profiles indicated P. salina desaturation conversions occurred with greater than 95% efficiency. Real-Time PCR revealed that expression of PsD8Des was higher than for the other two genes under normal growth conditions, while PsD5Des had the lowest expression level. The deduced amino acid sequences from all three genes contained three conserved histidine boxes and a cytochrome b(5) domain. Sequence alignment showed that the three genes were homologous to corresponding desaturases from other microalgae and fungi. The predicted activities of these three front-end desaturases leading to the synthesis of LC-PUFA were also confirmed in yeast and in higher plants.     

Studies on structure-function relationships of human choriogonadotropins with C-terminally shortened α-subunits. II. Stimulation of adenylate cyclase activity and depletion of cytochromeP-450 in the rat testis

Human choriogonadotropin (hCG) analogues, containing the native β-subunit and α-subunits enzymatically shortened by 2–3 amino acid residues, were used for studying influence of hCG on the content of microsomal progesterone-binding cytochromeP-450 in rat tests. When 2–3 residues have been renuwed from the α-subunit, the ability of the hormone analogue to stimulate adenylate cyclase of isolated rat Leydig cells was diminished by 55%. When the hCG analogue containing a des-(88–92)-α chain was applied, the residual activity of the adenylate cyclase was negligible. 18 h after administration to rats in vivo, the hormone species containing des-(Lys-91-Ser-92)-α or des-(90–92)-α, respectively, were found to have induced a decrease in microsomal cytochromeP-450 content with an effectiveness corresponding to their ability of stimulating the adenylate cyclase in vitro. However, when assayed 48 h after application, the desensitization of the microsomal cytochromeP-450 system had persisted in case of the hCG species containing a des-(90–92)-α chain but not in case of hCG consisting of des-(Lys-91-Ser-92)-α and a native β-subunit. From these results, it is concluded that short-term effects of hCG on the microsomal content of progesterone-binding cytochromeP-450 are mediated by the stimulation of adenylate cyclase. In contrast, the long-lasting action of hCG on this system seems not to be exclusively mediated by the increase in intracellular cAMP.     

Active metabolism of arachidonic acid by Kaposi sarcoma cells cultured from lung biopsies (KS-3); identification by HPLC and MS/MS of the predominant metaboilte secreted as the 11,12-epoxy-eicosatrienoic acid

The development of long-term culture of AIDS-KS cells has allowed us to investigate further a possible vascular origin of Kaposi sarcoma. Taking into account the relative specificity of arachidonic acid (AA) metabolism according to cell type, the AA ‘cascade’ was analyzed in cultured KS-3 cells established from lung biopsies and compared to human umbilical venous endothelial (H-UVE) cells and human myometrial smooth muscle (H-MSM) cells, under basal conditions and after stimulation with vasoactive agents such as histamine or thrombin. Considering strictly the ‘prostaglandin’ profile given by RIAs, the metabolism of AA was closer, whilst not identical, to H-UVE than to H-MSM cells. However, evaluation of all the eicosanoids released from [³H]AA labeled KS-3 cells revealed that the predominant metabolite was not prostacyclin (PGI₂), as suggested from PG RIAs, but an epoxy-eicosatrienoic acid (EET), identified as the 11, 12 isomer by HPLC and MS/MS. The synthesis of this EET is probably cytochrome P-450 monooxygenase dependent. Its potential role in the development of the KS tumor cells is under investigation.     

The effect of redox potential on the kinetics of fluorescence induction in Photosystem II particles from Phormidium laminosum. Sigmoidicity,energy transfer and the slow phase

Fluorescence induction curves in 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU)-inhibited Photosystem (PS) II particles isolated from the blue-green alga Phormidium laminosum have been analysed as a function of redox potential. Redox titration of the initial fluorescence indicated a single component with E_m,7.5 = +30 mV (n = 1) (Bowes, J., Horton, P. and Bendall, D.S. (1981) FEBS Lett. 135, 261–264). Despite this simplified electron acceptor system and the small number of chlorophylls per reaction centre, a sigmoidal induction curve was nevertheless seen. Sigmoidicity decreased as Q was reduced potentiometrically prior to induction such that the induction was exponential when the ratio $\text{F}{}_{\mathrm{<mtext>i</mtext>}}\text{F}{}_{\mathrm{<mtext>m</mtext>}}\text{= 0.64}$. These particles also showed a slow (β) phase of induction which titrated with an E_m value slightly more positive than that of the major quencher. It is concluded that the sigmoidal shape of the fluorescence induction curve observed in Phormidium PS II particles is not a consequence of a requirement for two photons to close the PS II reaction centre, but is generated as a result of energy transfer between photosynthetic units comprising one reaction centre per approx. 50 chlorophylls. Also, the existence of PS II heterogeneity (PS II_α, PS II_β centres) does not require a structurally differentiated chloroplast, but may only indicate the extent of aggregation of PS II centres.