Prenatal diagnosis of familial amyloidotic polyneuropathy: evidence for an early expression of the associated transthyretin methionine 30

Summary Transthyretin methionine 30 (TTR Met 30), which is associated with familial amyloidotic polyneuropathy, originates in a single base substitution (A for G) in the second exon of the TTR gene. This autosomal dominant disease can be diagnosed by RFLP analysis of NsiI-digested DNA. The amplification of DNA by PCR improves the diagnosis method, making it suitable for prenatal diagnosis. Using PCR-amplified DNA, prenatal diagnosis of two at-risk fetuses was performed. Control Met 30 and normal DNA (either genomic or produced by site directed mutagenesis) were processed in parallel. The diagnosis was made by hybridization with allele-specific oligonucleotide probes, and later confirmed by screening of the mutant protein in the amniotic fluid and, when possible, in the sera from the newborns. TTR Met 30 was detected in the amniotic fluid of a positive fetus whose father was the carrier of the mutation. This indicates that the mutant protein is expressed very early in development.     

Oxytocin enhances the basal release of uterine prostaglandin F2 alpha, but not that of PGE1, or of PGE2, and changes the metabolism of exogenous arachidonate, favouring the formation of prostaglandin F2 alpha and 5-HETE. Relationships with its uterotonic action and modulation by estradiol

We attempted to explore possible mechanism(s) subserving the influence of oxytocin on uterine motility by studying the action of the hormone on: 1) the contractile activity of isolated rat uteri in the presence or absence of indomethacin; 2) the synthesis and release of prostaglandins (PGs) into the solution incubating the uterine tissue as well as the metabolism of labelled arachidonic acid; 3) the uptake of 45Ca2+ by uterine strips. The experiments were bone with uterine preparations isolated from spayed rats treated or not with 17-beta-estradiol. The values of isometric developed tension (IDT) and of frequency of contractions (FC) induced by oxytocin in uterine strips isolated from spayed and spayed-estrogenized rats, were not modified by indomethacin at 10(-6) M. On the other hand, uterine strips from untreated spayed rats, release into the incubating medium approximately equal amounts of PGE1, PGE2 and PGF2 alpha. The in vitro presence of oxytocin (50 mU/ml) increased significantly (p 0.05) the output of PGF 2 alpha without changing the release of PGE1 or PGE2. Uteri from spayed rats injected prior to sacrifice with 17-beta-estradiol released significantly less PGE1 and PGE2 (p less than 0.005) than preparations from non-injected animals, whereas the output of PGF2 alpha in the suspending solution remained unchanged. Following estrogenization the addition of oxytocin to preparations obtained from spayed-estrogenized rats also increased the output of uterine PGF2 alpha (p less than 0.001) without changing that of PGs E1 or E2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of ova within rat oviducts on spontaneous motility and on prostaglandin production

The present study was performed in order to explore the influence of ova present within rat oviducts on: a) tubal spontaneous motility and b) oviduct prostaglandin production. It was found that the isometric developed tension (IDT) of tubes isolated from proestrous rats (preovulatory oviducts) was significantly higher (P less than 0.01) than the IDT of tubes from rats at estrus and at metestrus (postovulatory oviducts). After flushing the oviducts with KRB solution (i.e., after removing existing ova) the IDT of the oviducts obtained from estrous rats increased significantly (P less than 0.01), whereas the IDT of tubes isolated from proestrous rats (i.e., preparations without ova) was not modified. On the other hand, isolated tubes containing their corresponding ova released into the suspending solution significantly more PGE1 than PGE2 or PGF2 alpha (P less than 0.005). It was particularly interesting to find that after flushing the oviducts, tissue production of PGE1, PGE2 and PGF2 alpha was similar. Finally, when dose response curves for PGE1 and for PGE2 on the spontaneous contractions of oviducts isolated from rats at proestrus, estrus and metestrus were constructed, both PGs evoked an inhibitory inotropic action. The ED50 for PGE1 in tubes from estrous rats was significantly smaller (P less than 0.01) than that for metestrous animals but significantly greater (P less than 0.01) than that observed in oviducts from proestrous rats. The ED50 for PGE2 did not change in the different tested periods of the sex cycle. Results reported herein suggest the possibility that the ova present within rat oviducts, may influence their own transport along the tubes by modifying the amount of prostaglandins produced by the oviducts or via their own prostaglandin synthesis.     

A novel enhancing effect of platelet activating factor (PAF) on glucose oxidation in uteri from pregnant rats. Participation of prostaglandins and leukotrienes

The effects of platelet activating factor (PAF) on glucose oxidation in uterine strips isolated from rats in the 4 th and 5 th day of pregnancy, were explored. PAF, at a concentration of 10(-10) and 10(-8) M, augmented significantly the generation of 14CO2 from labelled glucose in uteri from pregnant rats in the 4 th day of pregnancy. When the tissue was obtained from 5 days pregnant rats, the addition of PAF at 10(-8) increased significantly more than PAF at 10(-10) M the metabolism of glucose. On the other hand, PAF at 10(-8) M failed to alter the uterine basal production of 14CO2 from labelled glucose in animals at estrus. BN52021, a specific PAF antagonist employed at 10(-5) M, blocked completely the action of PAF in the pregnant rat uterus. PGE1, PGE2 and PGF2 alpha enhanced significantly the formation of 14CO2 from labelled glucose in uteri from 5 days pregnant rats. Indomethacin, a well known inhibitor of prostaglandin synthesis, did not alter the basal glucose metabolism in uteri from 5 days pregnant rats, but antagonized completely the stimulating action of PAF on 14CO2 production from labelled glucose an effect that was partially reverted by the addition of PGE1, PGE2 or PGF2 alpha (10(-7) M). Furthermore, nordihydroguaiaretic acid (NDHGA), a specific inhibitor of 5-lipoxygenase at 10(-5) M, as well as FPL-55712, an antagonist of leukotrienes (LTs), at the same concentration, blocked the action of PAF on the metabolism of glucose. The action of NDHGA was partially counteracted by the addition of LTC4 at 10(-7) M.(ABSTRACT TRUNCATED AT 250 WORDS)     

Histopathology of experimental Aspergillus fumigatus keratitis

Histopathological studies in rabbit's eyes, 7 and 14 days after intracorneal inoculation with 1×10⁵ Aspergillus fumigatus conidia have been performed.Similar lesions were found in both periods with fungal hyphae in the anterior third of corneal stroma, round cell infiltration from the sclero-corneal edge and in the anterior chamber and, neovascularization.No lesions were found in the Descemet's membrane.Gomori silver-methenamine stain with hematoxiline-eosine counter-stain was found to be the most reliable stain to detect fungal presence in corneal stroma, and Masson's trichromic stain in the study of pathological changes in ocular elements.     

In vivo inhibition of nitrogenase by hydroxylamine in Rhodospirillaceae Role of nitric oxide

Rhodobacter capsulatus strains E1F1 and B10 and Rhodobacter sphaeroides DSM 158 did not use hydroxylamine as nitrogen source for growth but metabolized it mainly through the glutamine synthetase reaction. Hydroxylamine had a high toxicity for cells growing either under phototrophic or dark-aerobic conditions. l-methionine-d,l-sulfoximine partially inhibited hydroxylamine uptake and increased the inhibition time of nitrogenase activity by this nitrogen compound. Nitric oxide was also a powerful inhibitor of nitrogenase in intact cells of R. capsulatus. Since low amounts of NO were produced from hydroxylamine, short-term inhibition of nitrogenase in the presence of this compound could be mediated in vivo by nitric oxide.Abbreviations GS glutamine synthetase - MSX l-methionine-d,l-sulfoximine - MTA mixed alkyltrimethylammonium bromide     

The role of NADPH in the regulation of glucose-6-phosphate and 6-phosphogluconate dehydrogenases in rat adipose tissue

Summary Previous studies examining the regulation of the synthesis of G6PDH and 6PGDH in rat liver and adipose tissue have focused on the induction of these enzymes by different diets and some hormones. In rat liver these enzymatic activities seem to be regulated by a mechanism involving changes in the NADPH requirements. In this paper we have studied the effect of changes in the flux through different NADPH-consuming pathways on G6PDH and 6PGDH levels in adipose tissue and on the NADPH/NADP ratio. The results show that: I) an increase in the consumption of NADPH, caused by the activation of either fatty acid synthesis or detoxification systems which consume NADPH, is paralleled by an increase in the levels of these enzymes; II) when the increase in consumption of NADPH is prevented, the G6PDH and 6PGDH levels do not change.Abbreviations G6PDH Glucose-6-Phosphate Dehydrogenase - 6PGDH 6-Phosphogluconate Dehydrogenase - GR Glutathione Reductase - ME Malic Enzyme - tBHP t-Butyl Hydroperoxide - NF Nitrofurantoin - CumOOH Cumene Hydroperoxide     

Influence of Calcium Ion on Ethanol Tolerance of Saccharomyces bayanus and Alcoholic Fermentation by Yeasts

The addition of Ca²⁺ (as CaCl₂) in optimal concentrations (0.75 to 2.0 mM) to a fermentation medium with a trace contaminating concentration of Ca²⁺ (0.025 mM) led to the rapid production of higher concentrations of ethanol by Saccharomyces cerevisiae, Saccharomyces bayanus, and Kluyveromyces marxianus. The positive effect of calcium supplementation (0.75 mM) on alcoholic fermentation by S. bayanus was explained by the increase in its ethanol tolerance. The ethanol inhibition of growth and fermentation followed the equation μ_xi = μ_oi [1 - (X/X_mi)]ⁿⁱ, where μ_oi and μ_xi are, respectively, the specific growth (i = g) and fermentation (i = f) rates in the absence or presence of a concentration (X) of added ethanol, and X_mi is the maximal concentration of ethanol which allows growth or fermentation. The toxic power is given by n_i. In Ca²⁺ - supplemented medium (0.75 mM), n_g = 0.42 for growth and n_f = 0.43 for fermentation compared with 0.52 and 0.55, respectively, in unsupplemented medium; for both media, X_mg = 10% (vol/vol) and X_mf = 13% (vol/vol). For lethal concentrations of ethanol, the specific death rates were minimal for cells that were grown and incubated with ethanol in medium with an optimal concentration of Ca²⁺, maximal for cells grown and incubated with ethanol in unsupplemented medium, and intermediate for cells grown in unsupplemented medium and incubated with ethanol in calcium-supplemented medium. The effect of Ca²⁺ on the acidification curve of energized cells in the presence of ethanol was found to be closely associated with its protective effect on growth, fermentation, and viability.     

Modulation of the serotonin S2-receptor in brain after chronic lithium

In vivo effects of chronic lithium administration on dopaminergic and serotonergic receptor binding were studied in the striatum and cerebral cortex of the rat. [³H]Domperidone was used as the ligand for the dopaminergic receptor, and [³H]ketanserin for the serotonergic system. Long-term ingestion of lithium (2–3 months) resulted in high levels of lithium in the cerebral cortex and significantly higher potassium levels; the sodium content remained at normal levels. The kinetic constants (K _d andB _max) of [³H]domperidone binding sites measured in the striatum did not show any deviation from control values, but the receptor concentration (B _max) of [³H]ketanserin binding sites was significantly reduced in the cerebral cortex of lithium-treated rats. The apparent dissociation constant (K _d) was not changed. The results indicate that the serotonergic component of the [³H]spiperone binding site, which we had previously found to be affected by chronic lithium treatment and which was shown by Peroutka and Snyder (1) to be the 5-HT₂ receptor, is selectively affected by lithium.Special Issue dedicated to Prof. Eduardo De Robertis.     

Prostacyclin induces a surprising long-lasting motility in quiescent uterine strips (indomethacin-treated) isolated from ovariectomized rats

Dose-response curves for several prostaglandins (PGI2; PGD2; PGF2 and PGE2); BaCl2 or prostaglandin metabolites (15-keto-PGF2 alpha; 13,14-diOH-15-keto-PGF2 alpha; 6-keto-PGF1 alpha and 6-keto PGE1 in quiescent (indomethacin-treated) uterine strips from ovariectomized rats, were constructed. All PGs tested as well as BaCl2, triggered at different concentrations, evident phasic contractions. Within the range of concentrations tested the portion of the curves for the metabolites of PGF2 alpha was shifted to the right of that for PGF2 alpha itself; the curve for 6-keto-PGF1 alpha was displaced to the right of the curve for PGI2 and that for 6-keto-PGE1 to the left. It was also demonstrated that the uterine motility elicited by 10(-5) M PGF2 alpha and its metabolites was long lasting (more than 3 hours) and so it was the activity evoked by PGI2;6-keto-PGF1 alpha and BaCl2, but not the contractions following 6-keto-PGE1, which disappeared much earlier. The contractile tension after PGF2 alpha; 15-keto-PGF2 alpha; 13,14-diOH-15-keto-PGF2 alpha and PGI2, increased as time progressed whilst that evoked by 6-keto-PGF1 alpha or BaCl2 fluctuated during the same period around more constant levels. The surprising sustained and gradually increasing contractile activity after a single dose of an unstable prostaglandin such as PGI2, on the isolated rat uterus rendered quiescent by indomethacin, is discussed in terms of an effect associated to its transformation into more stable metabolites (6-keto-PGF1 alpha, or another not tested) or as a consequence of a factor which might protects prostacyclin from inactivation.