Isolation and sequence determination of cDNA encoding the major structural protein of human peripheral myelin.

A full length cDNA of the major structural protein of peripheral myelin (P0 protein) has been isolated from a cDNA library of human fetus spinal cord. The clone is 1948 base pairs (bp) in length and contains a 744 bp open reading frame encoding a polypeptide of 248 residues including 29 signal peptide. The deduced amino acid sequence is highly homologous to P0 protein from other species.     

Structural Effects on Arthrobacter Methylene Hydroxylase Activity

Arthrobacter 4-44-2 (ATCC 25581), capable of subterminal oxidation of n-hexadecane to 2-, 3-, and 4-alcoholic and ketonic products, was examined for the ability of this methylene hydroxylase capability to be induced and repressed and for structural relationships influencing methylene function oxidation. Induction was best carried out by use of n-alkanes from 10 to 16 carbons in length and was especially strong with methylcyclohexane among cyclic compounds tested. Induction was not observed with several related alcohols, 1-unsaturated compounds, or methoxy and ethoxy compounds tested. After induction, n-alkanes 14 and 16 carbons in length were transformed to the corresponding internal oxidation products; however, no activity was observed with even-carbon alkanes of shorter chain length. Hexadecene-1 and all alcohols tested, including cyclododecanol, were transformed to corresponding ketonic or aldehydic products. Cyclic compounds tested, including cyclododecane, were not oxidized by induced cells, suggesting that a methyl group plays a role in orientation of the substrate for the methylene hydroxylation but that the methyl function was not as critical after completion of the hydroxylation step regardless of structural configuration. Acetate strongly repressed induction of n-hexadecane methylene hydroxylase activity. Inducibility of methylene hydroxylase activity was confirmed by use of cell-free systems with methylcyclohexane as an inducer. A stimulation of methylene hydroxylase activity by addition of reduced pyridine nucleotides and ferrous ion was indicated.     

Pharmacological investigation of the mechanisms of platelet-activating factor induced mortality in the mouse

Although the platelets the mouse are refractory to the direct effects of platelet-activating-factor (PAF0, tail vein injection of 10–150 μg/kg PAF produces lethal anaphylatic shook. Sensitivity varies with strain and source: Swiss Webster mice show a range of sensitivity and DBA/2 (complement C5-deficient) mice are very resistant. At lethal doses within 15–45 min. Dexamethasone administered at least 1.5 hr prior consistently protects, whereas the cyclooxygenase inhibitors do not. Antihistamines, adrenergic antagonists, and methysergide have no effect, but cyproheptidine is partially protective at near lethal doses. Calcium entry blockers and calcium chelators, tetracyline and chlortetracycline are partially protective at very high doses consistent with non-specific effects on calcium dependent processes. The arachidonic acid lipoxygenase inhibitors BW755c, phenidone, nordihydroguai aretic acid and diphenyl disulfide provide nearly complete protection after oral administration of 50–200 mg/kg. Phosphodiesterase inhibitors and dapsone are also effectively orally. The luekotriene antagonist FPL55712 administered intraperitoneally (10 mg/kg) 5 min. prior to PAF challenge provides almost complete protection. PAF-induced mortality in the mouse represents a small animal model of systematic anaphylaxis particularly useful for the systematic testing of arachidonic acid lipoxygenase inhibitors and leukotriene antagonists.     

Retinoblastoma protein promotes uterine epithelial cell cycle arrest and necroptosis for embryo invasion

Retinoblastoma protein (RB) encoded by Rb1 is a prominent inducer of cell cycle arrest (CCA). The hormone progesterone (P₄) promotes CCA in the uterine epithelium and previous studies indicated that P₄ activates RB by reducing the phosphorylated, inactive form of RB. Here, we show that embryo implantation is impaired in uterine‐specific Rb1 knockout mice. We observe persistent cell proliferation of the Rb1‐deficient uterine epithelium until embryo attachment, loss of epithelial necroptosis, and trophoblast phagocytosis, which correlates with subsequent embryo invasion failure, indicating that Rb1‐induced CCA and necroptosis of uterine epithelium are involved in embryo invasion. Pre‐implantation P₄ supplementation is sufficient to restore these defects and embryo invasion. In Rb1‐deficient uterine epithelial cells, TNFα‐primed necroptosis is impaired, which is rescued by the treatment with a CCA inducer thymidine or P₄ through the upregulation of TNF receptor type 2. TNFα is expressed in the luminal epithelium and the embryo at the embryo attachment site. These results provide evidence that uterine Rb1‐induced CCA is involved in TNFα‐primed epithelial necroptosis at the implantation site for successful embryo invasion.     

Cyanogenic glucosides in grapevine: polymorphism, identification and developmental patterns

Twelve grapevine (Vitis vinifera L.) cultivars were surveyed for 'cyanide potential' (i.e. the total cyanide measured in beta-glucosidase-treated crude, boiled tissue extract) in mature leaves. Two related cultivars (Carignan and Ruby Cabernet) had mean cyanide potential (equivalent to 110 mgHCNkg-1fr.wt) ca. 25-fold greater than that of the other 10 cultivars, and so the trait is polymorphic in the species. In boiled leaf extracts of Carignan and Ruby Cabernet, free cyanide constituted a negligible fraction of the total cyanide potential because beta-glucosidase treatment was required to liberate the major cyanide fraction - which is therefore bound in glucosylated cyanogenic compound(s) (or cyanogenic glucosides). In addition, cyanide was liberated from ground leaf tissue of Ruby Cabernet but not Sultana (a cultivar with low cyanide potential). Hence, the high cyanide potential in Ruby Cabernet leaves is coupled with endogenous beta-glucosidase(s) activity and this cultivar may be considered 'cyanogenic'. A method was developed to detect and identify cyanogenic glucosides using liquid chromatography combined with tandem mass spectrometry (LC-MS/MS). Two putative cyanogenic glucosides were found in extracts from leaves of Carignan and Ruby Cabernet and were identified as the epimers prunasin and sambunigrin. Cyanide potential measured at three times over the growing season in young and mature leaves, petioles, tendrils, flowers, berries, seeds and roots of Ruby Cabernet was substantially higher in the leaves compared with all other tissues. This characterisation of cyanogenic glucoside accumulation in grapevine provides a basis for gauging the involvement of the trait in interactions of the species with its pests and pathogens.     

Aqueous flare elevation induced by transcorneal application of highly selective agonists for prostaglandin E2 receptor subtypes in pigmented rabbits: effect of tetramethylpyrazine.

We examined the disruptive effect of highly selective agonists for prostaglandin E2 receptor subtypes (EP1, EP2, EP3 and EP4) on the blood-aqueous barrier, and evaluated the inhibitory effect of tetramethylpyrazine, an active component of Ligusticum wallichii, on the elevation of aqueous flare induced by the EP agonists in pigmented rabbits. Highly selective EP agonists (ONO-DI-004, EP1 agonist; ONO-AE1-259-01, EP2 agonist; ONO-AE-248, EP3 agonist; ONO-AE1-329, EP4 agonist) at 12.5 to 250 microg/ml were transcorneally administered to the eyes of pigmented rabbits using a glass cylinder. Animals were pretreated intravenously with tetramethylpyrazine (10 or 30 mg/kg) 30 minutes before application of the EP2 or the EP4 agonist. Aqueous flare was measured using a laser flare-cell meter. Aqueous flare intensity was expressed as the area under the curve (AUC) in arbitrary units. After administration of ONO-AE1-259-01 or ONO-AE1-329, aqueous flare increased and then gradually decreased. ONO-DI-004 and ONO-AE-248 had almost no effect on aqueous flare elevation. The AUC of eyes in rabbits pretreated with tetramethylpyrazine, 10 or 30 mg/kg i.v., was significantly smaller than that of eyes in rabbits treated with ONO-AEI-259-01 alone. The AUC of eyes in rabbits pretreated with tetramethylpyrazine, 10 or 30 mg/kg i.v., was not significantly smaller than that of eyes in rabbits treated with ONO-AEI-329 only. The results indicated that EP2 and EP4 agonists induced aqueous flare elevation in pigmented rabbits, and that tetramethylpyrazine inhibited the aqueous flare elevation induced by the EP2 agonist but did not suppress the elevation induced by the EP4 agonist.     

Histological characteristics of the pelage skin of rough fur mice (C3H/HeJ- ruf/ruf).

Pelage skin of C3H/HeJ mice homozygous at an autosomal recessive mutant locus, rough fur (ruf) which is located on chromosome 9, was histologically analyzed. Sebaceous glands synthesizing lipids were larger in the mutant mice than in controls in an examination by Sudan IV staining. Electron microscopic analysis of the sebaceous gland showed that lipid droplets were denser in mutant mice than in control mice, and that they were irregular in shape in ruf mice while those of controls were round. Our results suggested that rough fur (ruf) mice might be an animal model for hyperlipogenesis of the pelage skin.     

Biosynthesis of avenic acid A in oat cv. Onward: studies with 14C or 15N labeled compounds

The precursory role of avenic acid A (AVA) in the biosynthesis of the mugineic acid family (MAs) of phytosiderophores was studied by feeding ¹⁴C or ¹⁵N labeled compounds into iron-deficient oat roots (Avena sativa L. cv. Onward). Carbon-14 of methionine was incorporated into AVA and 2-deoxymugineic acid (DMA) in the oat roots, while ¹⁴C of homoserine was not incorporated into either AVA or DMA. The molar radioactivity of DMA was higher than that of AVA. Incorporation of ¹⁵N into MAs was examined by feeding ¹⁵N-ammonium sulfate into oat roots. The value of ¹⁵N atom-% excess of DMA was higher than that of AVA.These results indicate that methionine, rather than homoserine, is the direct precursor of MAs in oat, which is similar to that in barley, and that AVA is not the precursor of the other MAs.     

Identification of the mutations in the T-protein gene causing typical and atypical nonketotic hyperglycinemia

We have investigated the molecular lesions of T-protein deficiency causing typical or atypical nonketotic hyperglycinemia (NKH) in two unrelated pedigrees. A patient with typical NKH was identified as being homozygous for a missense mutation in the T-protein gene, a G-to-A transition leading to a Gly-to-Asp substitution at amino acid 269 (G269D). Sibling patients of a second family with atypical NKH had two different missense mutations in the T-protein gene (compound heterozygote), a G-to-A transition leading to a Gly-to-Arg substitution at amino acid 47 (G47R) in one allele, and a G-to-A transition leading to an Arg-to-His substitution at amino acid 320 (R320H) in the other allele. Gly 269 is conserved in T-proteins of various species, even in E. coli, whereas Gly 47 and Arg 320 are replaced by Ala and Leu, respectively, in E. coli. The mutation occurring in more conservative amino acid residues thus results in more deleterious damage to the T-protein, and gives the severe clinical phenotype, viz., typical NKH.