Purification of the paraflagellar rod of the trypanosomatid Herpetomonas megaseliae and identification of some of its minor components

The paraflagellar rod (PFR) is a component of the flagellar cytoskeleton of trypanosomatid protozoa, representing a filamentous structure that runs alongside the common 9 + 2 microtubular axoneme. The high degree of ultrastructural complexity and organization of the PFR suggests that it might be formed by numerous biochemical components. However, biochemical analysis of the PFR has revealed, to date, a modest degree of complexity in what concerns both major and minor PFR proteins. In this paper the preparation of purified PFR fractions by a combination of conventional cell-fractionation procedures, non-ionic detergent treatment and limited proteolysis is described. Comparative SDS-PAGE analysis of the different purification steps indicates that the purified PFR fractions possess high amounts of the well-known major PFR proteins (77 and 83 kDa). Also, bands of 147, 139, 129 and 122 kDa are clearly enriched in such fractions and may correspond to minor PFR components. A slight enrichment in a specific fraction of a doublet of bands of 181/188 kDa suggest the participation of these proteins in the composition of the bridges between the PFR and the axoneme.     

Anti-platelet autoantibodies from ITP patients recognize an epitope in GPIIb/IIIa deduced by complementary hydropathy.

Idiopathic thrombocytopenic purpura (ITP) is a frequent platelet disorder due to the presence of anti-platelet autoantibodies. Recently a fibronectin/fibrinogen receptor in platelets, integrin GPIIb/IIIa, has been implicated as the antigen in chronic ITP. To examine the epitopes involved in the autoimmune response against GPIIb/IIIa we have used concepts from the complementary hydropathy principle. We used the peptide Trp-Thr-Val-Pro-Thr-Ala, WTVPTA (deduced from the complementary nucleotide sequence to that which codes for the Arg-Gly-Asp, RGD, domain in fibronectin), to test the immunologic activity of ITP sera. Sera from 31 patients with clinically defined ITP were tested in ELISA for reactivity towards WTVPTA and affinity purified GPIIb/IIIa. Seventeen sera (57%) reacted strongly with the glycoprotein complex, five of which reacted with the peptide. By affinity chromatography of one of these sera, we were able to show that antibodies that bind to the peptide are within the population that binds to GPIIb/IIIa. Liquid phase competition experiments revealed that binding of ITP serum to WTVPTA was inhibited only by a hydropathically compatible peptide. Our data indicate that autoantibodies can bind to hydropathically generated antigenic determinants and thus, render these peptides clinically important as diagnostic tools.     

Intracellular lectin-binding sites in symbiont-bearingCrithidia species

Crithidia oncopelti, C. deanei, andC. desouzai are flagellates of the Trypanosomatidae family that present bacterium-like endosymbionts in their cytoplasm. Direct and indirect lectin-gold labeling techniques were used at the electron microscopic level in Lowicryl K4M-embedded cells to demonstrate the presence of intracellular lectin-binding sites. We used the lectinsUlex europaeus I, Griffonia simplicifolia II, Ricinus communis I, Arachis hypogaea, G. simplicifolia I, Wistaria floribunda, Limulus polyphemus, andCanavalia ensiformis, which recognize -l-fucose, - and -N-acetylglucosamine, -galactose and -N-acetylgalactosamine, -galactose, -galactose, -N-acetylgalactosamine, sialic acid and -d-mannose, and -d-glucose residues, respectively. The nucleus was the cellular structure most frequently labeled by the lectins. The Golgi complex was seldom labeled, whereas the endoplasmic reticulum and the flagellar pocket presented a large number of binding sites. Symbionts had their two unit membranes weakly labeled by the different lectins but displayed no labeling of the space between the membranes.     

Characterization of the hamster FancG/Xrcc9 gene and mutations in CHO UV40 and NM3

The human FANCG/XRCC9 gene, which is defective in Fanconi anemia complementation group G (FA-G) cells, was first cloned by genetic complementation of the mitomycin C (MMC) sensitivity of CHO mutant UV40. The CHO NM3 mutant was subsequently assigned to the same complementation group. The parental AA8 CHO cells are hemizygous at the FancG locus, and we identified frameshift mutations that result in N-terminal truncations of the protein in both UV40 and NM3. Hypersensitivity to DNA cross-linking agents, such as MMC, typically characterizes FA cells. By introducing the native CHO FancG gene into mutant NM3, we demonstrate that hamster FancG fully corrects the 3-fold sensitivity to methyl methanesulfonate (MMS) as well as the 10-fold sensitivity to MMC, whereas resistance to ionizing radiation did not increase appreciably. In contrast, hamster cDNA transformants showed incomplete correction for both MMC and MMS sensitivity. The constitutively expressed FancG protein is present in the cytoplasmic, nuclear and chromatin fractions. FancG protein levels and subcellular localization do not change appreciably as a function of cell cycle position. Our results are consistent with roles of FancG in both the nuclear and cytoplasmic compartments to maintain genomic stability in response to various genotoxic agents.     

The pharmacology of the behavioral and hypothermic responses of rats to 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT)

The hypothermic and motor behavioural responses to 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) have been investigated in the rat. The dose-effect relationship showed that hypothermia appeared at a lower dose than a definite motor syndrome. The hypothermic response to 8-OH-DPAT was attenuated following depletion of 5-hydroxytryptamine (5-HT) by repeated intraperitoneal (IP) administration of parachlorophenylalanine (200 mg/kg) or by injection of 5,7-dihydroxytryptamine (5,7-DHT, 100 g) into the region of the third ventricle; the motor behavioural response produced simultaneously was not. Indeed, after 5,7-DHT, it was increased. Quipazine (1 mg/kg, IP) antagonised the hypothermic response and facilitated the motor behaviour. Clenbuterol (2.5 mg/kg, IP) increased both hypothermic and motor responses. (±)-propranolol was without effect on the simple hypermotility produced by 8-OH-DPAT, although it is known to antagonise the hypothermic and stereotyped motor responses. It is concluded that 8-OH-DPAT probably produces its hypothermic effects by actions at 5-HT receptors located presynaptically on 5-HT neurones, while the stereotyped components of the serotonin syndrome appear to be mediated by post-synaptic receptors.     

The effects of a 5-HT1 receptor ligand isapirone (TVX Q 7821) on 5-HT synthesis and the behavioural effects of 5-HT agonists in mice and rats

The effects of 2-(4-(4-(2-pyrimidinyl)-1-piperazinyl)-butyl)-1,2-benzoisothiazol-3(2H)one-1,1-dioxide hydrochloride (isapirone, TVX Q 7821), a putative 5-HT₁ receptor antagonist, has been studied on various models of 5-HT receptor sub-type function. In mice TVX Q 7821 produced a dose-dependent inhibition of the hypothermia induced by 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) with an ED₅₀ of 5.3 mg/kg suggesting that TVX Q 7821 was an antagonist of the presynaptic (possibly somato-dendritic) 5-HT_1A receptor. TVX Q 7821 did not alter the locomotor response to the suggested 5-HT_1B agonist RU 24969. The rate of mouse brain 5-HT synthesis was accelerated by TVX Q 7821 (10 mg/kg). 5-HT₂ receptor-mediated head twitch behaviour induced by precursor loading with 5-HTP was unaffected by TVX Q 7821 (10 mg/kg) pretreatment 75 min earlier, but the head-twitch induced by the agonist 5-methoxy-N,N-dimethyltryptamine was enhanced by prior treatment with TVX Q 7821.In rats the hypothermia induced by 8-OH-DPAT was partially antagonised by TVX Q 7821 while the behavioural serotonin syndrome induced by 8-OH-DPAT (a possible post-synaptic 5-HT_1B-mediated effect) was unaffected by TVX Q 7821 as was the locomotion induced by RU 24969.The data suggest that TVX Q 7821 is a good presynaptic 5-HT_1A antagonist in mice, as indicated by the 8-OH-DPAT-induced hypothermia and 5-HT synthesis rate studies. It did not antagonise 5-HT_1B-mediated behaviour in mice or rats and appeared to have an antagonist action at pre- but not post-synaptic 5-HT_1A receptors in rats. Offprint requests to: G.M. Goodwin     

Dietary Patterns During Pregnancy and Their Association with Gestational Weight Gain and Anthropometric Measurements at Birth

Maternal and Child Health Journal - Dietary patterns can influence maternal and child health outcomes. The study aims to characterize dietary patterns during pregnancy as well as to identify their...     

Vitamin A Status Improvement in Obesity: Findings and Perspectives Using Encapsulation Techniques

The association between obesity and vitamin A has been studied. Some studies point to the anti-obesity activity related to this vitamin, carotenoids with provitamin A activity, and carotenoid conversion products. This performance has been evaluated in respect of adipogenesis, metabolic activity, oxidation processes, secretory function, and oxidative stress modulation, showing a new property attributed to vitamin A in preventing and treating obesity. However, vitamin A and its precursors are highly sensitive and easily degraded when subjected to heat, the presence of light, and oxygen, in addition to losses related to the processes of digestion and absorption. In this context, encapsulation presents itself as an alternative capable of increasing vitamin A’s stability in the face of unfavorable conditions in the environment, which can reduce its functionality. Considering that vitamin A’s status shows a strong correlation with obesity and is an innovative theme, this article addresses the associations between vitamin A’s consumption and its precursors, encapsulated or not, and its physiological effects on obesity. The present narrative review points out those recent studies that demonstrate that vitamin A and its encapsulated precursors have the most preserved functionality, which guarantees better effects on obesity therapy.