Vincanorin und Vincarein,zwei neue Alkaloide ausVinca minor L.

Summary The isolation of two new alkaloids fromVinca minor L. (Apocynaceae) is described. Vincarein, C₂₁H₂₄N₂O₄, and Vincanorin, C₁₉H₂₂N₂O, both probably belong to the group of indol alkaloids.     

Composition of epicuticular wax of rice,Oryza sativa

Summary The epicuticular wax of rice, varietyRibe, comprised n-alkanes, esters, aldehydes and free alcohols. The nalkanes contained 4 major chain lengths, C₂₇, C₂₉, C₃₁ and C₃₃. Triacontanal and dotriacontanal were the major aldehydes. Octacosanol comprised 89% of the free alcohols. The esters were mainly esters of C₁₆ to C₂₄ acids with C₂₂ to C₃₀ alcohols.This research was supported by the Consiglio Nazionale delle Ricerche, Rome.     

Protease-activated-receptor-2 affects protease-activated-receptor-1-driven breast cancer

Mammalian protease-activated-receptor-1 and -2 (PAR₁ and PAR₂) are activated by proteases found in the flexible microenvironment of a tumor and play a central role in breast cancer. We propose in the present study that PAR₁ and PAR₂ act together as a functional unit during malignant and physiological invasion processes. This notion is supported by assessing pro-tumor functions in the presence of short hairpin; shRNA knocked-down hPar2 or by the use of a truncated PAR₂ devoid of the entire cytoplasmic tail. Silencing of hPar2 by shRNA-attenuated thrombin induced PAR₁ signaling as recapitulated by inhibiting the assembly of Etk/Bmx or Akt onto PAR₁-C-tail, by thrombin-instigated colony formation and invasion. Strikingly, shRNA-hPar2 also inhibited the TFLLRN selective PAR₁ pro-tumor functions. In addition, while evaluating the physiological invasion process of placenta extravillous trophoblast (EVT) organ culture, we observed inhibition of both thrombin or the selective PAR₁ ligand; TFLLRNPNDK induced EVT invasion by shRNA-hPar2 but not by scrambled shRNA-hPar2. In parallel, when a truncated PAR₂ was utilized in a xenograft mouse model, it inhibited PAR₁–PAR₂-driven tumor growth in vivo. Similarly, it also attenuated the interaction of Etk/Bmx with the PAR₁-C-tail in vitro and decreased markedly selective PAR₁-induced Matrigel invasion. Confocal images demonstrated co-localization of PAR₁ and PAR₂ in HEK293T cells over-expressing YFP-hPar2 and HA-hPar1. Co-immuno-precipitation analyses revealed PAR₁-PAR₂ complex formation but no PAR₁-CXCR4 complex was formed. Taken together, our observations show that PAR₁ and PAR₂ act as a functional unit in tumor development and placenta-uterus interactions. This conclusion may have significant consequences on future breast cancer therapeutic modalities and improved late pregnancy outcome.     

Molecular compounds without chemical bonding

Zusammenfassung Die Infrarotspektren der Einschlussverbindungen [Ni(CN)₂(NH₃)(C₆H₆)] und [Ni(CN)₂(NH₃)C₄H₅N] wurden untersucht und mit den Spektren von gasförmigen und flüssigen C₆H₆ bzw. C₄H₆N verglichen.

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Clathrate work was carried out under a research grant by University of Western Australia. Nickel cyanide-ammonia clathrates were further investigated by the author at the Punjab University (Chandigarh, India), University of Rome (Rome, Italy) and Fisk University (Nashville, Tennessee, USA).     

Production and characterization of antibodies cross-reactive with major aflatoxins

Antibodies cross-reactive with 4 major aflatoxins were demonstrated three weeks after immunization of rabbits with an immunogen which was prepared by conjugating aflatoxin B₃ to bovine serum albumin. Aflatoxin B₃ was first converted to its hemisuccinate before conjugation to the protein. Tritiated aflatoxin B₁ (AFB₁) was used as the marker ligand both for antibody titer determination as well as for analysis of antibody specificity. Competitive RIA revealed that the antibodies have good cross-reactivity with aflatoxins B₁, B₂, G₁, and G₂ when tritiated AFB₁ was used as the marker ligand. The concentrations causing 50% inhibition of binding of³H-AFB₁ to the antibodies by unlabeled aflatoxins B₁, B₂, G₁, G₂ and B₃ were found to be 0.25, 3.34, 0.32, 4.0 and 0.53 ng/assay, respectively. The antibodies could be used for simultaneous analysis of aflatoxins B₁ and G₁, two of the most important toxic metabolites produced byAspergillus flavus andA. parasiticus.     

Ion-solvent interaction from viscosity,apparent molar volume and conductivity data of bromates,iodates and sulphates of potassium and sodium in dioxane-water mixtures at different temperatures

Summary The viscosity, apparent molar volume and conductivity of KBrO₃, NaBrO₃, KIO₃, NaIO₃, K₂SO₄ and Na₂SO₄ at mass fraction of dioxane (10, 20 and 30%) — water mixtures at 30–45°C±0.01°C have been measured. The ions appear to interact and the ion-solvent interaction is of the order BrO ₃ ^– >IO ₃ ^– >SO ₄ ^2– .     

Specific immunosuppression of IgE response to hapten DNP by DNP linked to monoclonal IgG1 in rats

Summary The induction of the anti-DNP IgE in rat was suppressed by pretreatment of rats with the tolerogen synthesized by coupling DNP to rat IgG, i.e.; DNP_7–10-IgG. It was found that DNP₁₀-IgG₁ was an effective tolerogen, whereas other DNP conjugates, i.e. DNP₉-IgM, DNP₉-IgA, DNP₁₀-IgE, DNP₁₀-IgG_2c and DNP₁₀-IgG_2a were ineffective.This work was partly supported by a research grant from the Medical Council of Canada to W.Y.L.     

Functional and biochemical characteristics of mitochondrial fractions from rat liver in cold-induced oxidative stress

We determined characteristics of rat liver mitochondrial fractions, resolved at 1000 (M₁), 3000 (M₃), and 10,000 g (M₁₀) after 2 and 10 days cold exposure. In all groups, the M₁ fraction exhibited the highest oxidative capacity, oxidative damage, H₂O₂ production rate, and susceptibility to stress conditions, and the lowest antioxidant levels. Cold exposure increased cytochrome oxidase activity in all fractions and succinate-supported O₂ consumption in the M₁ and M₁₀ fractions during state 3 and state 4 respiration, respectively. With succinate, the H₂O₂ release rate increased in all fractions during state 4 and state 3 respiration, whereas with pyruvate/malate, it increased only during state 4 respiration. Increases in tissue mitochondrial proteins caused a faster H₂O₂ flow from the mitochondrial to cytosolic compartment, which was limited by the reduction in the M₁ fraction. Despite increased liposoluble antioxidant levels, cold also caused enhanced oxidative damage and susceptibility to oxidative challenge and Ca²⁺-induced swelling in all fractions. These changes leading to elimination of H₂O₂-overproducing mitochondria avoided excessive tissue damage. We propose that triiodothyronine, whose levels increase in the cold environment, brings about the biochemical changes producing oxidative damage and those limiting its extent.Received 16 July 2004; received after revision 27 September 2004; accepted 18 October 2004     

Role of the ubiquitin–proteasome system in the regulation of P2Y13 receptor expression: impact on hepatic HDL uptake

The protective effect of high density lipoproteins (HDL) against atherosclerosis is mainly attributed to their capacity to transport excess cholesterol from peripheral tissues back to the liver for further elimination into the bile, a process called reverse cholesterol transport (RCT). Recently, the importance of the P2Y₁₃ receptor (P2Y₁₃-R) was highlighted in HDL metabolism since HDL uptake by the liver was decreased in P2Y₁₃-R deficient mice, which translated into impaired RCT. Here, we investigated for the first time the molecular mechanisms regulating cell surface expression of P2Y₁₃-R. When transiently expressed, P2Y₁₃-R was mainly detected in the endoplasmic reticulum (ER) and strongly subjected to proteasome degradation while its homologous P2Y₁₂ receptor (P2Y₁₂-R) was efficiently targeted to the plasma membrane. We observed an inverse correlation between cell surface expression and ubiquitination level of P2Y₁₃-R in the ER, suggesting a close link between ubiquitination of P2Y₁₃-R and its efficient targeting to the plasma membrane. The C-terminus tail exchange between P2Y₁₃-R and P2Y₁₂-R strongly restored plasma membrane expression of P2Y₁₃-R, suggesting the involvement of the intra-cytoplasmic tail of P2Y₁₃-R in expression defect. Accordingly, proteasomal inhibition increased plasma membrane expression of functionally active P2Y₁₃-R in hepatocytes, and consequently stimulated P2Y₁₃-R-mediated HDL endocytosis. Importantly, proteasomal inhibition strongly potentiated HDL hepatic uptake (>200 %) in wild-type but not in P2Y₁₃-R-deficient mice, thus reinforcing the role of P2Y₁₃-R expression in regulating HDL metabolism. Therefore, specific inhibition of the ubiquitin–proteasome system might be a novel powerful HDL therapy to enhance P2Y₁₃-R expression and consequently promote the overall RCT.     

Participation of ACTH1–10 and ACTH4–10 on the melatonin modulation of benzodiazepine receptors in rat cerebral cortex

In this study, we examined the effect of intracerebroventricular (i.c.v) injection of melatonin and/or ACTH_1–10 and ACTH_4–10 on [³H]flunitrazepam binding sites in the cerebral cortex of hypophysectomized rats. Hypophysectomy increased the B_max (maximum number of binding sites) of benzodiazepine (BNZ) receptors for at least 7 days after surgery, without changing K_D (dissociation constant). The i.c.v. injection of melatonin to hypophysectomized rats significantly increased B_max, whereas the same doses of melatonin were ineffective in sham-operated animals. In both cases, K_D values were unchanged. The i.c.v injection of ACTH_1–10 to hypophysectomized animals significantly increased B_max, an effect that was enhanced by simultaneous i.c.v. injection of ACTH_1–10+melatonin, reaching higher values of B_max than the i.c.v. injection of these hormones individually. No significant changes in K_D values were found after ACTH_1–10 and/or melatonin administration. However, the i.c.v. injection of ACTH_4–10 to hypophysectomized rats did not change B_max, although it significantly increased K_D values, indicating a decrease in the BNZ binding affinity. Melatonin injection counteracted this effect of ACTH_4–10, returning K_D to the control value. Moreover, although the lower dose of i.c.v. melatonin used, 10 ng, was unable to modify B_max of BNZ binding in the ACTH_4–10-injected group, the higher dose, 20 ng, significantly increased B_max. The results suggest that these ACTH-derived peptides can modulate the effect of melatonin on brain benzodiazepine receptors.     

Effects of pCai and pHi on cell-to-cell coupling

Summary Internal longitudinal resistance (r_i), a determinant of cardiac conduction, is affected by changes in intracellular calcium and protons. However, the role and mechanism by which H⁺ and Ca²⁺ may modulate r_i is uncertain. Cable analysis was performed in cardiac Purkinje fibers to measure r_i during various interventions. In some experiments, intracellular pH (pH_i) was recorded simultaneously to study the pH_i-r_i relation. Both intracellular Ca²⁺ and H⁺ independently modified r_i. However, internal resistance of cardiac fibers was insensitive to pH_i changes compared to other tissues. A latent period preceded the pH_i-related changes in r_i and the amount of change depended upon methodology. The results suggest that direct action of protons on r_i may be subordinate to other regulatory processes. Ionic regulation of internal longitudinal resistance may occur by more than one mechanism: i) direct cationic binding to sites on junctional membrane proteins; and ii) H⁺- or Ca²⁺-dependent phosphorylation of junctional proteins.     

The determination of volatile aldehydes in gases and vapours

Zusammenfassung Es wird eine neue Methode vorgeschlagen, Acetaldehyd, Propionaldehyd und Butyraldehyd in Gegenwart von CH₄, C₂H₆, C₃H₈, C₂H₄, C₃H₆, C₂H₂, CO₂, CO, N₂, H₂ und O₂ quantitativ zu bestimmen. Die Methode besteht darin, das Gasgemenge mit Hilfe eines Orsatschen Apparates durch eine bei 20°C gesättigte, Chromtrioxyd und Schwefelsäure enthaltende Natriumsulfatlösung zu schicken, welche die oben genannten Aldehyde selektiv absorbiert.     

Specificity in the hydrolysis of N-acyl-L-phenylalanine 4-nitroanilides by chymotrypsin

Summary The affinity of N-acyl-L-phenylalanine 4-nitroanilides for chymotrypsin is enhanced as the hydrophobicity of non-amino acid residues in the P₂-position of the substrates increases, whereas k_cat remains nearly constant. On the other hand, if alanine or leucine is in the P₂-position k_cat increases with decreasing K_M.     

Comparative potency of gibberellins in inducing parthenocarpic fruit growth inMalus sylvestris mill

Zusammenfassung Die biologische Wirksamkeit der Gibberelline A₁ bis A₁₀, A₁₃ und A₁₄ wurde bei der Einsetzung von Parthenokarpie inMalus sylvestris Mill. festgestellt. Die Gibberelline A₄ und A₇ waren sehr wirksam, A₁, A₂, A₃, A₉, A₁₀, A₁₃ und A₁₄ waren von mittelmässiger Wirksamkeit, während A₅, A₆ und A₈ waren unwirksam. Die mehr wirksamen Gibberelline besitzen, vom molekularen Standpunkt betrachtet, in der Stellung 7 keine OH-Gruppe.

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On leave from Osaka Prefecture University, Osaka, Japan.     

Die Dünnschichtchromatographie von Gibberellinen

Summary Thin-layer chromatography on silica gel is described as a new method for the separation and identification of the gibberellins A₁, A₃, A₄, A₅, A₇, A₈, and A₉.     

Changes in the T4/T3 molar ratio following thyrotrophin releasing hormone injection in cattle

Summary The injection of thyrotropin releasing hormone into cattle resulted in a rapid decrease in the T₄/T₃ molar ratio. 2 breeds of cattle, Shorthorn and Africander Cross were studied. The decrease in the T₄/T₃ molar ratio was significantly greater in the Shorthorn breed. It is concluded that acute stimulation of the thyroid gland with TRH results in enhanced release of both T₃ and T₄ and that T₃ is discharged more rapidly than T₄.     

Hydrogen peroxide protects tobacco from oxidative stress by inducing a set of antioxidant enzymes

Tolerance against oxidative stress generated by high light intensities or the catalase inhibitor aminotriazole (AT) was induced in intact tobacco plants by spraying them with hydrogen peroxide (H₂O₂). Stress tolerance was concomitant with an enhanced antioxidant status as reflected by higher activity and/or protein levels of catalase, ascorbate peroxidase, guaiacol peroxidases, and glutathione peroxidase, as well as an increased glutathione pool. The induced stress tolerance was dependent on the dose of H₂O₂ applied. Moderate doses of H₂O₂ enhanced the antioxidant status and induced stress tolerance, while higher concentrations caused oxidative stress and symptoms resembling a hypersensitive response. In stress-tolerant plants, induction of catalase was 1.5-fold, that of ascorbate peroxidase and glutathione peroxidase was 2-fold, and that of guaiacol peroxidases was approximately 3-fold. Stress resistance was monitored by measuring levels of malondialdehyde, an indicator of lipid peroxidation. The levels of malondialdehyde in all H₂O₂-treated plants exposed to subsequent high light or AT stress were similar to those of unstressed plants, whereas lipid peroxidation in H₂O₂-untreated plants stressed with either high light or AT was 1.5- or 2-fold higher, respectively. Although all stress factors caused increases in the levels of reduced glutathione, its levels were much higher in all H₂O₂-pretreated plants. Moreover, significant accumulation of oxidized glutathione was observed only in plants that were not pretreated with H₂O₂. Extending the AT stress period from 1 to 7 days resulted in death of tobacco plants that were not pretreated with H₂O₂, while all H₂O₂-pretreated plants remained little affected by the prolonged treatment. Thus, activation of the plant antioxidant system by H₂O₂ plays an important role in the induced tolerance against oxidative stress. Received 11 December 2001; received after revision 25 January 2002; accepted 4 February 2002     

Autophagic activity in cortical neurons under acute oxidative stress directly contributes to cell death

Primary neurons undergo insult-dependent programmed cell death. We examined autophagy as a process contributing to cell death in cortical neurons after treatment with either hydrogen peroxide (H₂O₂) or staurosporine. Although caspase-9 activation and cleavage of procaspase-3 were significant following staurosporine treatment, neither was observed following H₂O₂ treatment, indicating a non-apoptotic death. Autophagic activity increased rapidly with H₂O₂, but slowly with staurosporine, as quantified by processing of endogenous LC3. Autophagic induction by both stressors increased the abundance of fluorescent puncta formed by GFP-LC3, which could be blocked by 3-methyladenine. Significantly, such inhibition of autophagy blocked cell death induced by H₂O₂ but not staurosporine. Suppression of Atg7 inhibited cell death by H₂O₂, but not staurosporine, whereas suppression of Beclin 1 prevented cell death by both treatments, suggesting it has a complex role regulating both apoptosis and autophagy. We conclude that autophagic mechanisms are activated in an insult-dependent manner and that H₂O₂ induces autophagic cell death.