Adjuvant chemotherapy for patients with gastric neuroendocrine carcinomas or mixed adenoneuroendocrine carcinomas.

BACKGROUND: The aim of this study was to evaluate whether adjuvant chemotherapy is associated with improved survival in patients with resectable gastric neuroendocrine carcinomas (G-NECs) or mixed adenoneuroendocrine carcinomas (G-MANECs). METHODS: The study included patients with G-NECs or G-MANECs who underwent surgery in one of 21 centres in China between 2004 and 2016. Propensity score matching analysis was used to reduce selection bias, and overall survival (OS) in different treatment groups was estimated by the Kaplan-Meier method. RESULTS: In total, 804 patients with resectable G-NECs or G-MANECs were included, of whom 490 (60·9 per cent) received adjuvant chemotherapy. After propensity score matching, OS in the chemotherapy group was similar to that in the no-chemotherapy group. Among patients with G-NECs, survival in the fluorouracil (5-FU)-based chemotherapy group and the non-5-FU-based chemotherapy group was similar to that in the no-chemotherapy group. Similarly, etoposide plus cisplatin or irinotecan plus cisplatin was not associated with better OS in patients with G-NECs. Among patients with G-MANECs, OS in the non-5-FU-based chemotherapy group was worse than that in the no-chemotherapy group. Patients with G-MANECs did not have better OS when platinum-based chemotherapy was used. CONCLUSION: There was no survival benefit in patients who received adjuvant chemotherapy for G-NECs or G-MANECs.

ANTECEDENTES: El objetivo de este estudio fue evaluar si la quimioterapia adyuvante mejoraba la supervivencia en pacientes con carcinomas gástricos resecables neuroendocrinos (gastric neuroendocrine carcinomas, G-NECs) y carcinomas adenoneuroendocrinos mixtos (mixed adenoneuroendocrine carcinomas, G-MANECs). MÉTODOS: Se incluyeron pacientes con G-NECs y G-MANECs tratados quirúrgicamente en 21 centros en China entre 2004 y 2016. Se utilizó un análisis de emparejamiento por puntaje de propensión para reducir el sesgo de selección y el método de Kaplan-Meier para estimar la supervivencia global (overall survival, OS) de los pacientes en los diferentes grupos de tratamiento. RESULTADOS: En total, se incluyeron en el estudio 804 pacientes con G-NECs y G-MANECs resecables y 490 pacientes (60,9%) recibieron quimioterapia adyuvante. Después del emparejamiento por puntaje de propensión, la OS del grupo con quimioterapia fue similar a la del grupo sin quimioterapia. En los pacientes con G-NECs, la supervivencia en los grupos con quimioterapia basada en 5-FU (fluorouracilo) y de quimioterapia sin 5-FU fue similar a la del grupo sin quimioterapia. Asimismo, la combinación de etopósido y cisplatino o de irinotecán y cisplatino no se asoció con una mejor OS en pacientes con G-NECs. En pacientes con G-MANECs, la OS del grupo con quimioterapia sin 5-FU fue peor que la del grupo sin quimioterapia. Los pacientes con G-MANECs no presentaron una mejor OS cuando se administró quimioterapia basada en platinos. CONCLUSIÓN: La administración de quimioterapia adyuvante en pacientes con G-NECs y G-MANECs no mejoró la supervivencia.

[Aluminuminduced impairment in primary cultured rat choroid plexus epithelial cells].

OBJECTIVE: To investigate the impairment in primary cultured rat choroid plexus epithelial cells (CPECs)induced by aluminum. METHODS: The choroid plexus isolated from Sprague-Dawley rats 14 days old was cut into pieces and digested by trypsin in the sterile area. The obtained single cells were cultured in DMEM with 1% epidermal growth factor and 20% fetal calf serum. Five days later, immunohistochemistry with anti-transthyretin antibody was used to identify the purity of cultured cells. The well-grown cells were treated with aluminum lactate at different concentrations (0, 100, 400, and 1 600 µmol/L for control, lowdose, mediumdose, and highdose groups). Fortyeight hours later, the cell viability, apoptotic rate, level of reactive oxygen species (ROS), and activity of superoxide dismutase (SOD)were measured in each group to evaluate the impairment in primary cultured rat CPECs by aluminum. RESULTS: More than 95% of the cultured cells were identified as CPECs. The medium-and high-dose groups had significantly lower cell viability than the control group(86.74%±4.03% vs 100%, P<0.01; 81.90%±9.17% vs 100%, P<0.01). The high-dose group had significantly lower cell viability than the lowdose group (81.90%±9.17% vs 92.92%±8.81%, P<0.01). The medium-and high-dose groups had significantly higher apoptotic rates than the control group (7.26%±0.99% vs 1.29%±0.03%, P<0.01; 22.25%±1.55% vs 1.29%±0.03%, P<0.01)and the low-dose group (7.26%±0.99% vs 1.68%±0.27%, P<0.01; 22.25%±1.55% vs 1.68%±0.27%, P<0.01). The high-dose group had a significantly higher apoptotic rate than the medium-dose group (22.25%±1.55% vs 7.26%±0.99%, P<0.01). The mediumand high-dose groups had significantly higher fluorescence intensity of ROS than the control group (22.23%±0.41% vs 17.24%±0.09%, P<0.05; 25.10%±1.13% vs 17.24%±0.09%, P<0.05)and the lowdose group (22.23%±0.41% vs 18.31%±0.21%, P<0.05; 25.10%±1.13% vs 18.31%±0.21%, P<0.05). The highdose group had significantly higher fluorescence intensity of ROS than the mediumdose group (25.10%±1.13% vs 22.23%±0.41%, P< 0.05). The low-, medium-and high-dose groups had significantly lower SOD activity than the control group[(28.65±0.74)U/g Hb vs (37.35±1.05)U/g Hb, P<0.05; (22.75±1.94)U/g Hb vs (37.35±1.05)U/g Hb, P<0.05; (13.29±0.64)U/g Hb vs(37.35±1.05)U/g Hb, P<0.05]. The medium-and high-dose groups had significantly lower SOD activity than the low-dose group[(22.75±1.94)U/g Hb vs(28.65±0.74)U/g Hb, P<0.05; (13.29±0.64)U/g Hb vs (28.65±0.74)U/g Hb, P<0.05], while the high-dose group had had significantly lower SOD activity than the medium-dose group[(13.29±0.64)U/g Hb vs (22.75±1.94)U/g Hb, P<0.05]. There were no significant differences in cell viability, apoptotic rate, level of ROS, or activity of SOD between any other two groups (P>0.05). CONCLUSION: Aluminum lactate may induce impairment in primary cultured rat CPECs. It reduces the cell viability, elevates the apoptotic rate, and causes oxidative stress.

Effects of maternal folic acid supplementation on gene methylation and being small for gestational age.

BACKGROUND: Being small for gestational age (SGA), a foetal growth abnormality, has a long-lasting impact on childhood health. Its aetiology and underlying mechanisms are not well understood. Underlying epigenetic changes of imprinted genes have emerged as a potential pathological pathway because they may be associated with growth, including SGA. As a common methyl donor, folic acid (FA) is essential for DNA methylation, synthesis and repair, and FA supplementation is widely recommended for women planning pregnancy. The present study aimed to investigate the inter-relationships among methylation levels of two imprinted genes [H19 differentially methylated regions (DMRs) and MEST DMRs], maternal FA supplementation and SGA. METHODS: We conducted a case-control study. Umbilical cord blood was taken from 39 SGA infants and 49 controls whose birth weights are appropriate for gestational age (AGA). DNA methylation levels of H19 and MEST DMRs were determined by an analysis of mass array quantitative methylation. RESULTS: Statistically significantly higher methylation levels were observed at sites 7.8, 9 and 17.18 of H19 (P = 0.030, 0.016 and 0.050, respectively) in the SGA infants compared to the AGA group. In addition, the association was stronger in male births where the mothers took FA around conception at six H19 sites (P = 0.004, 0.005, 0.048, 0.002, 0.021 and 0.005, respectively). CONCLUSIONS: Methylation levels at H19 DMRs were higher in SGA infants compared to AGA controls. It appears that the association may be influenced by maternal peri-conception FA supplementation and also be sex-specific.

Genetic polymorphisms and tissue expression of interleukin-22 associated with risk and therapeutic response of gastric mucosa-associated lymphoid tissue lymphoma.

Chronic Helicobacter pylori-stimulated immune reactions determine the pathogenesis of gastric mucosa-associated lymphoid tissue (MALT) lymphoma. We aimed to explore the genetic predisposition to this lymphoma and its clinical implication. A total of 68 patients and 140 unrelated controls were genotyped for 84 single-nucleotide polymorphisms in genes encoding cytokines, chemokines and related receptors that play important roles in T cell-mediated gastrointestinal immunity. Five genotypes in IL-22, namely CC at rs1179246, CC at rs2227485, AA at rs4913428, AA at rs1026788 and TT at rs7314777, were associated with disease susceptibility. The former four genotypes resided in the same linkage disequilibrium block (r(2)=0.99) that conferred an approximately threefold higher risk. In vitro experiments demonstrated that co-culturing peripheral mononuclear cells or CD4(+) T cells with H. pylori stimulated the secretion of interleukin-22 (IL-22), and that IL-22 induced the expression of antimicrobial proteins, RegIIIα and lipocalin-2, in gastric epithelial cells. Furthermore, patients with gastric tissue expressing IL-22 were more likely to respond to H. pylori eradication (14/22 vs 4/19, P<0.006). We conclude that susceptibility of gastric MALT lymphoma is influenced by genetic polymorphisms in IL-22, the product of which is involved in mucosal immunity against H. pylori and associated with tumor response to H. pylori eradication.

Chemical and biological assessment of angelica roots from different cultivated regions in a chinese herbal decoction danggui buxue tang.

Roots of Angelica sinensis (Danggui) have been used in promoting blood circulation as herbal medicine for over 2000 years in China. Another species of Angelica roots called A. gigas is being used in Korea. To reveal the efficiency of different Angelica roots, the chemical and biological properties of Angelica roots from different cultivated regions were compared. Roots of A. sinensis contained higher levels of ferulic acid, Z-ligustilide, and senkyunolide A, while high amounts of butylphthalide and Z-butylenephthalide were found in A. gigas roots. The extracts deriving from A. gigas roots showed better effects in osteogenic and estrogenic properties than that of A. sinensis from China. However, this difference was markedly reduced when the Angelica roots were being prepared in a Chinese herbal decoction together with Astragali Radix as Danggui Buxue Tang. In contrast, the herbal decoction prepared from A. sinensis roots showed better responses in cell cultures. In addition, the extracts of A. gigas roots showed strong cell toxicity both as single herb and as Danggui Buxue Tang. This result revealed the distinct properties of Angelica roots from China and Korea suggesting the specific usage of herb in preparing a unique herbal decoction.

Importance of wine-treated Angelica Sinensis Radix in Si Wu Tang, a traditional herbal formula for treating women's ailments.

Si Wu Tang (Four Agents Decoction), a traditional Chinese decoction composed of Angelica Sinensis Radix, Chuanxiong Rhizoma, Paeoniae Radix Alba, and Rehmanniae Radix Praeparata in a ratio of 1 : 1 : 1 : 1, has been used to treat women's diseases for more than a thousand years. According to the original description of Si Wu Tang, Angelica Sinensis Radix should be treated with wine. However, the importance of this wine-treated Angelica Sinensis Radix in Si Wu Tang's function has not been identified. In this article, the chemical and biological properties of two decoctions processed in different ways (Si Wu Tang with crude Angelica Sinensis Radix and Si Wu Tang with wine-treated Angelica Sinensis Radix) were compared for examination. The herbal decoction Si Wu Tang prepared from wine-treated Angelica Sinensis Radix contained much different amounts of its active compounds. Compared with Si Wu Tang using crude Angelica Sinensis Radix, Si Wu Tang prepared from wine-treated Angelica Sinensis Radix had better biological responses. Therefore, these findings accentuate the functional importance of herbs treated with wine in the Chinese decoction.

Application of a nanotechnology antimicrobial spray to prevent lower urinary tract infection: a multicenter urology trial.

BACKGROUND: Catheter-associated urinary tract infection (CAUTI) is a common nosocomial device-associated infection. It is now recognized that the high infection rates were caused by the formation of biofilm on the surface of the catheters that decreases the susceptibility to antibiotics and results in anti-microbial resistance.In this study, we performed an in vitro test to explore the mechanism of biofilm formation and subsequently conducted a multi-center clinical trial to investigate the efficacy of CAUTI prevention with the application of JUC, a nanotechnology antimicrobial spray. METHODS: Siliconized latex urinary catheters were cut into fragments and sterilized by autoclaving. The sterilized sample fragments were randomly divided into the therapy and control group, whereby they were sprayed with JUC and distilled water respectively and dried before use.The experimental standard strains of Escherichia coli (E. coli) were isolated from the urine samples of patients. At 16 hours and 7 days of incubation, the samples were extracted for confocal laser scanning microscopy.A total of 1,150 patients were accrued in the clinical study. Patients were randomized according to the order of surgical treatment. The odd array of patients was assigned as the therapy group (JUC), and the even array of patients was assigned as the control group (normal saline). RESULTS: After 16 hours of culture, bacterial biofilm formed on the surface of sample fragments from the control group. In the therapy group, no bacterial biofilm formation was observed on the sample fragments. No significant increase in bacterial colony count was observed in the therapy group after 7 days of incubation.On the 7th day of catheterization, urine samples were collected for bacterial culture before extubation. Significant difference was observed in the incidence of bacteriuria between the therapy group and control group (4.52% vs. 13.04%, p < 0.001). CONCLUSIONS: In this study, the effectiveness of JUC in preventing CAUTI in a hospital setting was demonstrated in both in vitro and clinical studies.

Genome affinity and meiotic behaviour in trigenomic hybrids and their doubled allohexaploids between three cultivated Brassica allotetraploids and Brassica fruticulosa.

The wild species Brassica fruticulosa Cyr. (FF, 2n = 16) is closely related to the cultivated Brassica species.Through interspecific reciprocal crosses between B. fruticulosa and three cultivated Brassica allotetraploids (AABB, AACC,and BBCC where A = 10, B = 8, and C = 9), four trigenomic hybrids (F.AC, 2n = 27; F.AB, 2n = 26; F.BC, 2n = 25;BC.F, 2n = 25) were produced. By chromosome doubling of respective hybrids, three allohexaploids (FF.AACC, 2n = 54;FF.AABB, 2n = 52; BBCC.FF, 2n = 50) were synthesized. In pollen mother cells (PMCs) of the trigenomic hybrids, 1­2 autosyndetic bivalents were detected within A, B, and C genomes but only one within F genome; 1­3 allosyndetic bivalents between any two genomes were observed, and a closer relationship of F and B genomes than F and A genomes or F and C genomes was revealed. The all ohexaploids showed a generally low but different pollen fertilities. The chromosomes in PMCs were predominantly paired as bivalents but some univalents and multivalents at variable frequencies were observed.The bivalents of homologous pairing for each genome prevailed, but all osyndetic quadrivalents and hexavalents involving any two genomes were observed, together with autosyndetic quadrivalents for A, B, and C genomes but not the F genome.The nondiploidized cytological behaviour of these allohexaploids contributed to their low fertility. The relationships between the genome affinity and meiotic behavior in these allohexaploids were discussed.

Active pharmaceutical ingredients and mechanisms underlying phasic myometrial contractions stimulated with the saponin extract from Paris polyphylla Sm. var. yunnanensis used for abnormal uterine bleeding.

BACKGROUND: Total steroidal saponins of Paris polyphylla Sm. var. yunnanensis (TSSP) have been widely used in China for the treatment of abnormal uterine bleeding (AUB). But until now, the main active constituents and the mechanisms underlying the pharmacological actions on uterine activity have not been described. METHODS: Total steroidal saponins were extracted with EtOH and purified by chromatography. In vitro isometric contraction studies were performed using myometrial strips from estrogen-primed or pregnant rats. Intracellular calcium was monitored under a confocal microscope using Fluo-3 AM-loaded myometrial cells. RESULTS: TSSP dose-dependently induced phasic myometrial contractions in vitro. Experiments with calcium channel blockers or kinase inhibitors demonstrated that the TSSP-stimulated myometrial contraction was mediated by an increase in [Ca(2+)](i) via influx of extracellular calcium and release of intracellular calcium. Through bioassay-guided separation, it was found that total spirostanol saponins exhibited contractile activity in myometrium and Pennogenin-3-O-alpha-L-arabinofuranosyl(1-->4)[alpha-L-rhamnopyranosyl(1-->2)]-beta-D-glucopyranoside (PARG) was identified as the active ingredient of TSSP. Furthermore, the contractile response of rat myometrium to PARG was significantly enhanced with advancing pregnancy. CONCLUSIONS: These data provide evidence that myometrial contractility stimulated by TSSP results from [Ca(2+)](i) increase and supports the possibility that some spirostanol gylcosides may represent a new type of contractile agonist for the uterus.

Effects of Shugansanjie Tang on matrix metalloproteinases 1, 3 and 9 and telomerase reverse transcriptase expression in human breast cells in vitro.

BACKGROUND: The traditional Chinese medication Shugansanjie Tang (SGT), whose active component is Akebia Trifoliate Koidz, possesses potential anti-tumor and immunostimulatory effects especially for breast cancer. The invasive processes of such cancers have been attributed to matrix metalloproteinases (MMPs) which may be the key factor mediating this process. Telomerase reverse transcriptase (TERT) catalyze the lengthening of telomeres, which prolongs cell life and interrupts natural cell death. The aim of this study is to determine the effects of Shugansanjie Tang on MMP levels and TERT activity using breast cancer cell lines. MATERIALS AND METHODS: We used the breast cancer cell lines MDA-MB-231 and BT-483. Cell inhibition rate was measured by WST-1 reagent, cell apoptosis by Apoptotic DNA Ladder Kit, mRNA expression of MMP-1, MMP-3 and MMP-9 by PCR and TERT by immunohistochemistry stain. RESULTS: Comparing to the control group, the test group showed lower cell growth rate, decreased mRNA expression of MMP-1, MMP-3 and MMP-9 production and less intense staining of MMPs with diaminobenzidine tetrahydrochloride. CONCLUSION: This study demonstrated that Shugansanjie Tang inhibits the growth of breast cancer cells by apoptosis and lowers the level of certain matrix metalloproteinases and activity of telomerase reverse transcriptase in breast cancer in vitro.

In vitro estrogenic activities of Chinese medicinal plants traditionally used for the management of menopausal symptoms.

The estrogenic activity of 70% EtOH extracts of 32 traditional Chinese medicinal plants, selected according to their reported efficacy for the treatment of menopausal symptoms, was assessed using a recombinant yeast system with both a human estrogen receptor expression plasmid and a reporter plasmid. Among them, 11 (34%) species proved to be active. Polygonum cuspidatum had the highest estrogenic relative potency (RP) (3.28 x 10(-3)), followed by Rheumpalmatum (3.85 x 10(-4)), Cassia obtusifolia (3.49 x 10(-4)), Polygonum multiflorum (2.87 x 10(-4)), Epimedium brevicornum (2.30 x 10(-4)), Psoralea corylifolia (1.90 x 10(-4)), Cynomorium songaricum (1.78 x 10(-4)), Belamcanda chinensis (1.26 x 10(-4)), Scutellaria baicalensis (8.77 x 10(-5)), Astragalus membranaceus (8.47 x 10(-5)) and Pueraria lobata (6.17 x 10(-5)). The EC(50) value of 17beta-estradiol used as the positive control was 0.205+/-0.025 ng/ml (RP=100). This study gave support to the reported efficacy of Chinese medicines used for hormone replacement therapy.

Further molecular characterisation of potyviruses infecting aroid plants for medicinal use in China.

Degenerate primers were used to detect and amplify 3'-terminal genome fragments of potyviruses from medicinal aroid plants growing at 16 sites in China. Virus was detected in 7 samples of which six, all of Pinellia ternata, contained a strain of soybean mosaic virus (SMV) similar to that previously reported from this host in China. The complete sequence of one isolate and the P1 protein coding region of the other isolates were also sequenced. In all cases, the P1 proteins resembled isolates of Dasheen mosaic virus (DsMV) more closely than SMV, confirming earlier suggestions of recombination in this region. In a phylogenetic analysis of SMV, DsMV and related sequences, the aroid sequences of SMV formed a distinct group which also included a sequence published as Zantedeschia symptomless virus (AF469171). One of the P. ternata samples was also infected with a second potyvirus, the 3'-terminal sequence of which was similar to DsMV and to some sequences published as Vanilla mosaic virus. The seventh infected sample was Typhonium flagelliforme and the virus from it was identified from its sequence as zantedeschia mosaic virus (ZaMV), providing the first report of this virus from mainland China.

Detection and classification of allexiviruses from garlic in China.

Degenerate primers for RT-PCR were designed and used to amplify genome fragments ( c. 750 nt in the coat protein-ORF6 region) of allexiviruses from a total of 28 garlic samples from 24 provinces in China. Many samples contained more than one distinct sequence. A total of 60 different sequences were obtained. Phylogenetic analysis and two-way comparisons were used to assess the status of the sequences and to re-examine the criteria for distinguishing species within the genus. Most of the sequences could be allocated to either Garlic virus D or Garlic virus X on the basis of sequence similarity but some appeared to be intermediate between existing species. There were no sequences of Garlic virus C or Shallot virus X. A comparison with the related genera Carlavirus, Foveavirus and Potexvirus suggests that the published allexivirus species demarcation criteria may have been drawn too tightly and should be re-examined.

Sequence analysis demonstrates that Onion yellow dwarf virus isolates from China contain a P3 region much larger than other potyviruses.

The complete sequence of an isolate of Onion yellow dwarf virus (OYDV) from Yuhang, Zhejiang province, China, was determined. It was 10538 nts in length and was predicted to encode a polyprotein 3403 amino acids (aa) long with a calculated M(r) of 385.1 kDa. The predicted P3 protein (530 aa) was larger than that of any of the potyviruses sequenced to date (344-378 aa). The additional sequence occurs at the N-terminus of the protein, does not represent a duplication from elsewhere in the OYDV genome and could not be matched to any other sequences in the databases. Similar sequences were found in 4 other Chinese OYDV isolates. Phylogenetic analysis of the amino acid sequences of the polyprotein showed that OYDV is distantly related to Pea seed-borne mosaic virus and the potyviruses of grasses and cereals.

Effect of trilinolein on superoxide dismutase activity and left ventricular pressure in isolated rat hearts subjected to hypoxia and normoxic perfusion.

Oxygen-derived free radicals have been implicated in the development of myocardial injury during hypoxia/reperfusion. Antioxidants can effectively inhibit the formation of free radicals and ameliorate the myocardial damage which may occur during hypoxia/reperfusion. Trilinolein is a triacylglycerol recently purified from the traditional Chinese medicinal plant Panax pseudo-ginseng. It has linoleic-acid residues as the only type of fatty acid residue in all three esterified positions of the triacyglycerol. It has been proposed that decreased endogenous superoxide dismutase (SOD) activity may contribute to free radical-mediated reperfusion injury of the ischemic myocardium. In the present study, when isolated rat hearts were subjected to hypoxia for 10, 30, 60 and 90 min without normoxic perfusion, a significant decrease in Mn-SOD activity was shown throughout the period of hypoxia, whereas the Cu.Zn-SOD activity was increased at 10 and 30 min but was not different from the baseline at 60 and 90 min of hypoxia. In rat hearts pretreated with 10(-7) mol/l trilinolein and subjected to 60 min of hypoxia without normoxic perfusion, Cu.Zn-SOD was augmented compared with baseline and compared with hearts subjected to 60 min of hypoxia without trilinolein, whereas Mn-SOD activity was still reduced compared with baseline, although less so than after 60 min of hypoxia without trilinolein. Pretreatment with trilinolein was associated with better preservation of left ventricular function during hypoxia and more rapid return to recovery during normoxic perfusion. This myocardial protective effect may be related to an antioxidant effect through potentiation of SOD, particularly Cu.Zn-SOD during hypoxia.

Myocardial protective effect of trilinolein: an antioxidant isolated from the medicinal plant Panax pseudoginseng.

In a previous study we demonstrated that trilinolein, a natural plant triacylglycerol, is a novel myocardial protective agent in vivo. The mechanism probably involves an antioxidant effect. This work investigated the mechanism of myocardial protection of trilinolein to determine if inhibition of calcium influx and alteration of activity of superoxide dismutase are involved. In isolated cardiomyocytes, pretreatment with trilinolein at a low concentration of 10(-9) M effectively reduced 45Ca2+ influx stimulated by hypoxia/normoxia by 34%. In isolated perfused rat heart subjected to 60 min global hypoxemia without reperfusion, pretreatment with 10(-7) M trilinolein for 15 min reduced infarct size by 37%. Assay of superoxide dismutase-mRNA by Northern blot analysis in in vivo rat heart subjected to 30 min ischaemia and 10 min reperfusion showed pretreatment with 10(-7) M trilinolein had a synergistic action with antioxidant systems preventing the rise in superoxide dismutase-mRNA. These results reconfirm the myocardial protection of trilinolein and suggest it may be related to antioxidant activity and inhibition of 45Ca2+ influx.

Synthesis and characterization of supramolecular protein aggregates: self-assembled, molecularly-ordered, tubes from electrostatic complementation of glutamine synthetase dodecamers.

Dodecameric Escherichia coli glutamine synthetase (GS) is formed from identical subunits arranged in face-to-face hexameric rings. In the presence of Zn2+ and other transition metal ions the individual dodecamers 'stack' to form protein tubes. Previous results have suggested that six binuclear intermolecular metal binding sites are generated at each dodecamer-dodecamer interface by juxtaposition of the N-terminal helices of each subunit adjacent to an analogous helix from a docked dodecamer. In principle, replacement of one of the metal binding sites within each pair of helices with charged amino acids could generate electrostatic interactions that would provide the basis for heterospecific protein-protein interactions. In turn, this would allow for ordered assembly of protein tubes with alternating, chemically distinguishable, components. This hypothesis was tested by replacement of one of the metalligating histidines (His12) with aspartic acid, arginine or cysteine. The H12C mutant was further elaborated by selective thiol modification, with either of the charged reagents 2-iodo-acetic acid or 2-chloro-acetamidine, which yield glutamate (H12C-IA) or arginine (H12C-CA) mimics at position 12. Light scattering and electron microscopy were used to monitor the 'stacking ability' of these variants in the presence of Zn2+. No, or few, GS 'tubes' were observed in solutions containing only H12D, H12R, H12C-CA or H12C-IA, in the presence or absence of Zn2+. In contrast, in mixtures containing H12C-CA and either H12D or H12C-IA, the complementary GS variants stack in the presence of 100 microM Zn2+, with apparent second order rate constants that are comparable to the wild type dodecamers. Fluorescence energy transfer experiments with fluorescein-labeled H12C-IA (donor) and rhodamine-labeled H12C-CA (acceptor) were performed and compared with the energy transfer efficiency with mixtures containing variable ratios of acceptor-labeled and donor-labeled wild type GS; the wild type mixtures provide a benchmark for the extent of energy transfer expected in random linear arrangements of donor and acceptor. The efficiency of metal-dependent energy transfer in mixtures containing the acceptor-labeled H12C-CA and the donor-labeled H12C-IA was 3.2-fold greater than expected for a random distribution of charged variants. Together, the results indicate that the charged variants provide a mechanism for heterospecific interaction between chemically distinguishable dodecamers that align in an ordered one-dimensional array.