Tumor immune-gene expression profiles and peripheral immune phenotypes associated with clinical outcomes of locally advanced pancreatic cancer following FOLFIRINOX.

BACKGROUND: A comprehensive analysis of peripheral immune cell phenotypes and tumor immune-gene expression profiles in locally advanced pancreatic cancer patients treated with neoadjuvant chemotherapy in a phase II clinical trial was carried out. METHODS: Patients were treated with neoadjuvant modified folinic acid, fluorouracil, irinotecan hydrochloride, oxaliplatin (mFOLFIRINOX) followed by surgery and adjuvant gemcitabine at the Asan Medical Center. Correlations between survival outcomes and baseline peripheral immune cells and their changes during preoperative chemotherapy were analyzed. Patients who had surgery were divided into two groups according to achievement of disease-free survival >10 months (achieved versus failed). Differential expression and pathway analysis of immune-related genes were carried out using the Nanostring platform, and immune cells within the tumor microenvironment were compared by immunohistochemistry. RESULTS: Forty-four patients were treated in the phase II clinical trial. Higher baseline CD14+CD11c+HLA-DR+ monocytes (P = 0.044) and lower Foxp3+CD4+ T cells (P = 0.02) were associated with poor progression-free survival of neoadjuvant mFOLFIRINOX. During the preoperative chemotherapy, PD-1 T cells significantly decreased (P = 0.0110). Differential expression and pathway analysis of immune-genes from the resected tumor after neoadjuvant treatment revealed transforming growth factor-ß pathway enrichment and higher expression of MARCO (adjusted P < 0.05) associated with early recurrence. Enrichment of the Th1 pathway and higher peritumoral CD8+ T cells (P = 0.0103) were associated with durable disease-free survival from surgery (>10 months) following neoadjuvant mFOLFIRINOX. CONCLUSIONS: Our results identify potential immune biomarkers for locally advanced pancreatic cancer and provide insights into pancreatic cancer immunity.

Label-free screening of single biomolecules through resistive pulse sensing technology for precision medicine applications.

Employing integrated nano- and microfluidic circuits for detecting and characterizing biological compounds through resistive pulse sensing technology is a vibrant area of research at the interface of biotechnology and nanotechnology. Resistive pulse sensing platforms can be customized to study virtually any particle of choice which can be threaded through a fluidic channel and enable label-free single-particle interrogation with the primary read-out signal being an electric current fingerprint. The ability to perform label-free molecular screening with single-molecule and even single binding site resolution makes resistive pulse sensing technology a powerful tool for analyzing the smallest units of biological systems and how they interact with each other on a molecular level. This task is at the core of experimental systems biology and in particular 'omics research which in combination with next-generation DNA-sequencing and next-generation drug discovery and design forms the foundation of a novel disruptive medical paradigm commonly referred to as personalized medicine or precision medicine. DNA-sequencing has approached the 1000-Dollar-Genome milestone allowing for decoding a complete human genome with unmatched speed and at low cost. Increased sequencing efficiency yields massive amounts of genomic data. Analyzing this data in combination with medical and biometric health data eventually enables understanding the pathways from individual genes to physiological functions. Access to this information triggers fundamental questions for doctors and patients alike: what are the chances of an outbreak for a specific disease? Can individual risks be managed and if so how? Which drugs are available and how should they be applied? Could a new drug be tailored to an individual's genetic predisposition fast and in an affordable way? In order to provide answers and real-life value to patients, the rapid evolvement of novel computing approaches for analyzing big data in systems genomics has to be accompanied by an equally strong effort to develop next-generation DNA-sequencing and next-generation drug screening and design platforms. In that context lab-on-a-chip devices utilizing nanopore- and nanochannel based resistive pulse-sensing technology for DNA-sequencing and protein screening applications occupy a key role. This paper describes the status quo of resistive pulse sensing technology for these two application areas with a special focus on current technology trends and challenges ahead.

The Effect of Plant Extracts on In-vitro Ruminal Fermentation, Methanogenesis and Methane-related Microbes in the Rumen.

The effect on methanogens attached to the surface of rumen ciliate protozoa by the addition of plant extracts (pine needles and ginkgo leaves) was studied with particular reference to their effectiveness for decreasing methane emission. The plant extracts (pine needles and ginkgo leaves) were added to an in vitro fermentation incubated with rumen fluid. The microbial population including bacteria, ciliated-associated methanogen, four different groups of methanogens and Fibrobacter succinogenes were quantified by using the real-time PCR. Gas profiles including methane, carbon dioxide and hydrogen, and runinal fermentation characteristics were observed in vitro. The methane emission from samples with an addition of individual juices from pine needles, ginkgo leaves and 70% ethanol extract from ginko leaves was significantly lower (p<0.05, 27.1, 28.1 and 28.1 vs 34.0 ml/g DM) than that of the control, respectively. Total VFAs in samples with an addition of any of the plant extracts were significantly lower than that of the control (p<0.05) as well. The order Methanococcales and the order Methanosarcinales were not detected by using PCR in any incubated mixtures. The ciliate-associated methanogens population decreased from 25% to 49% in the plant extacts as compared to control. We speculate that the supplementation of juice from pine needles and ginkgo leaves extract (70% ethanol extract) decreased the protozoa population resulting in a reduction of methane emission in the rumen and thus inhibiting methanogenesis. The order Methanobacteriales community was affected by addition of all plant extracts and decreased to less than the control, while the order Methanomicrobiales population showed an increase to more than that of the control. The F. succinogenes, the major fibrolytic microorganism, population in all added plant extracts was increased to greater than that of the control. In conclusion, pine needles and ginkgo leaves extracts appear to have properties that decrease methanogenesis by inhibiting protozoa species and may have a potential for use as additives for ruminants.

Effect of treatment with a mixture of bacteria and fibrolytic enzymes on the quality and safety of corn silage infested with different levels of rust.

This project aimed to determine if a dual-purpose bacterial inoculant could mitigate potential adverse effects of increasing levels of rust infestation on the quality, aerobic stability, and safety of corn silage. Corn plants with no rust infestation (NR), or medium (all leaves on the lower half of the plant affected, MR), or high (all leaves affected, HR) levels of southern rust infestation were harvested at random locations on a field, chopped, and ensiled without (control, CON) or with a dual-purpose inoculant applied at a rate that supplied 1×10(5) cfu/g of Pediococcus pentosaceus 12455 and 4×10(5) cfu/g of Lactobacillus buchneri 40788. Each treatment was prepared in quadruplicate in 20-L mini silos and ensiled for 97 d. As the level of rust infestation increased, the concentrations of dry matter (DM) and neutral detergent fiber increased, whereas DM digestibility decreased by up to 16%. Control HR silages also had lower 24-h neutral detergent fiber digestibility (NDFD; 36.2% of DM) than CON MR (39.8%) or NR silages (38.1%). Inoculation increased the NDFD of NR (43.4%) and MR silages (45.7%) but not HR silages (33.0%). Concentrations of lactate and volatile fatty acids decreased with increasing rust infestation in CON silages, but this trend was absent in inoculated silages. In HR silages, inoculation increased aerobic stability by 75% (77.3 vs. 44 h), and prevented production of aflatoxin (5.2 vs. 0 mg/kg). The concentration of aflatoxin in uninoculated HR silages exceeded action levels stipulated by the US Food and Drug Administration. In conclusion, increasing rust infestation was associated with reductions in the nutritive value and fermentation of corn silage. Inoculation reduced adverse effects of rust infestation on the fermentation, increased 24-h NDFD of NR and MR silages, and decreased aerobic spoilage and aflatoxin production in HR silages.

Effects of feeding crude glycerin on performance and ruminal kinetics of lactating Holstein cows fed corn silage- or cottonseed hull-based, low-fiber diets.

The objective was to determine whether crude glycerin could partially replace concentrate ingredients in corn silage- or cottonseed hull-based diets formulated to support minimal milk fat production without reducing milk production. Multiparous, lactating Holstein cows (n=24; 116 ± 13d in milk) were assigned to dietary treatments arranged in a 2 × 3 factorial design; namely, 2 dietary roughage sources (cottonseed hulls or corn silage) and 3 dietary concentrations of glycerin [0, 5, or 10% on a dry matter (DM) basis]. Four different cows received each dietary treatment in each of 3 periods such that each diet was evaluated using 12 cows. Crude glycerin, produced using soybean oil, contained 12% water, 5% oil, 6.8% sodium chloride, and 0.4% methanol. Glycerin partially replaced ground corn, corn gluten feed, and citrus pulp. Diets of minimum fiber concentrations were fed to lactating dairy cows and resulted in low concentrations of milk fat (averaging 3.12% for cows fed diets without glycerin). The effects of glycerin on cow performance and ruminal measurements were the same for both dietary roughage sources with the exception of feed efficiency. Replacing concentrate with crude glycerin at 5% of dietary DM increased DM intake without increasing milk yield. Concentration and yield of milk fat were reduced when glycerin was fed at 10% of dietary DM. This was accompanied by a 30% reduction in apparent total-tract digestion of dietary neutral detergent fiber. Crude glycerin affected the microbial population in the rumen as evidenced by increased molar proportions of propionic, butyric, and valeric acids and decreased molar proportions of acetic acid. Efficiency of N utilization was improved as evidenced by lower concentrations of blood urea nitrogen and ruminal ammonia-N. Cows fed cottonseed hull-based diets consumed 5.3 kg/d more DM but produced only 1.7 kg/d more milk, resulting in reduced efficiency. Increased production of ruminal microbial protein, molar proportion of propionic acid, and passage of ruminal fluid resulted from feeding the cottonseed hull- versus corn silage-based diets, although apparent digestibilities of DM and neutral detergent fiber were reduced. Replacing 5 and 10% of concentrate ingredients with crude glycerin improved efficiency of 4% fat-corrected milk production when corn silage-based diets were fed but decreased it when cottonseed hull-based diets were fed.

Effect of fibrolytic enzyme application to low- and high-concentrate diets on the performance of lactating dairy cattle.

The objective of this study was to examine the effect of applying a fibrolytic enzyme preparation to diets with high (48% of diet dry matter, DM) or low (33% of diet DM) proportions of concentrate on production performance of lactating dairy cows. Sixty lactating Holstein cows (589 kg ± 20; 22 ± 3 d in milk) were stratified according to milk production and parity and randomly assigned to 4 treatments with a 2 × 2 factorial arrangement. Dietary treatments included the following: 1) low-concentrate diet (LC); 2) LC plus enzyme (LCE); 3) high-concentrate diet (HC); and 4) HC plus enzyme (HCE). The enzyme was sprayed at a rate of 3.4 mg of enzyme/g of DM on the total mixed ration daily and the trial lasted for 63 d. A second experiment with a 4 × 4 Latin square design used 4 ruminally fistulated cows to measure treatment effects on ruminal fermentation and in situ ruminal dry matter degradation during four 18-d periods. Enzyme application did not affect dry matter intake (DMI; 23.9 vs. 22.3 kg/d) or milk production (32.8 vs. 34.2 kg/d) but decreased estimated CH(4) production, increased total volatile fatty acid concentration (114.5 vs. 125.7 mM), apparent total tract digestibility of DM (69.8 vs. 72.6%), crude protein (CP; 69.2 vs. 73.3%), acid detergent fiber (50.4 vs. 54.8%), neutral detergent fiber (53.7 vs. 55.4%), and the efficiency of milk production (1.44 vs. 1.60 kg of milk/kg of DMI). Feeding more concentrates increased DMI (21.5 vs. 24.8 kg/d), milk yield (32.2 vs. 34.7 kg/d), milk protein yield (0.89 vs. 0.99 kg/d), and DM (69.9 vs. 72.6%), but decreased ruminal pH (6.31 vs. 6.06). Compared with cows fed HC, those fed LCE had lower DMI (20.8 vs. 25.7 kg/d) and CP intake (3.9 vs. 4.8 kg/d), greater ruminal pH (6.36 vs. 6.10), and similar milk yield (33.2 ± 1.1 kg/d). Consequently, the efficiency of milk production was greater in cows fed LCE than those fed HC (1.69 vs. 1.42 kg of milk/kg of DMI). This fibrolytic enzyme increased the digestibility of DM, CP, neutral detergent fiber, and acid detergent fiber and the efficiency of milk production by dairy cows. Enzyme application to the low-concentrate diet resulted in as much milk production as that from cows fed the untreated high-concentrate diet.

Glutamine induces heat-shock protein-70 and glutathione expression and attenuates ischemic damage in rat islets.

BACKGROUND: The transplantation of isolated islets is believed to be an attractive approach for cure of diabetes mellitus. Heat-shock protein (HSP70), which plays a vital role in cellular protection, has been detected in various tissues subjected to stress. Glutamine (GLN) is an important cellular fuel and an essential precursor for the antioxidant glutathione (GSH). It is believed to enhance cellular survival against a variety of stressful stimuli through HSP70. Thus, we performed this study to examine the hypothesis that preoperative GLN administration induces HSP70 and GSH expression before islet transplantation attenuating ischemic damage to rat islets. METHODS: Adult male Sprague-Dawley (SD) rats were randomly divided into two groups according to the administration of GLN after islet isolation. Group A served as the controls, receiving no GLN. Group B islet cells were cultured with L-GLN (10 mmol/L) supplementation for 24 hours. The GSH levels were measured in islet cells. Both HSP70 and proteins related to apoptosis were analyzed in islet cells by Western blots. Isolated rat islets were cultured with interleukin (IL)-1beta. Nitrite production was measured using the Griess reagent. RESULTS: The GSH levels were significantly elevated in the glutamine-treated group. HSP70 expression in islets treated with GLN was markedly stronger compared with the control group. The basal Bcl-2 expression was markedly increased by GLN treatment. The GLN-treated group showed attenuated IL-1beta-induced injury in association with NO production. CONCLUSION: These results suggested that preoperative GLN administration induced HSP70 and GSH expressions before islet transplantation, thus attenuating IL-1beta-induced injury in association with NO production and apoptosis, which might be potential tool to mitigate the ischemic damage to islet cells and the early inflammation at the site of implantation through a self-protective mechanism.

Complete oxidation of volatile organic compounds over Ce/Cu/gamma-AL2O3 catalyst.

The effect of cerium (Ce) addition into Cu (5, 10 or 15 wt%)/gamma-Al2O3 catalysts on the catalyst properties and catalytic activity was investigated for the complete oxidation of volatile organic compounds (VOCs). X-ray diffraction (XRD), the Brunauer Emmett Teller method (BET), temperature programmed reduction (TPR) by H2, and N2O pulse titration were used to characterize a series of supported copper catalysts modified with cerium. Cerium was observed to be an inhibitor for 5 wt% and promoter for 10 or 15 wt% Cu/gamma-Al2O3 catalyst. The results of TPR, average crystallite size and dispersion indicated that even though Ce loadings on 10 and 15 wt% Cu/gamma-Al2O3 caused a reduction in BET surface area of the catalysts, the loaded amounts of Ce enhanced the catalytic activity through the formation of highly dispersed copper clusters. Kinetic parameters were developed for individual benzene, toluene and o-xylene (BTX) for 5 wt% Ce/10 wt% Cu/gamma-Al2O3 catalyst at temperatures ranging from 210 to 240 degrees C. The Mars and Van Krevelen model was found to be an adequate description of the catalytic oxidation of BTX for this study. The activity sequence with respect to the BTX molecules was found to be benzene > toluene > o-xylene under the surface-reaction-controlled region.

Effect of method of applying fibrolytic enzymes or ammonia to Bermudagrass hay on feed intake, digestion, and growth of beef steers.

This study examined how different methods of applying a fibrolytic enzyme or ammonia affect the nutritive value of Bermudagrass hay and the performance of beef cattle. Fifty Angus x Brangus crossbred steers (mean initial BW 244 +/- 26 kg) were individually fed for ad libitum intake of a 5-wk regrowth of a mixture of Florakirk and Tifton 44 Bermudagrass [Cynodon dactylon (L.) Pers] hay for 84 d with a concentrate supplement (77% soybean hull pellets, 23% cottonseed meal (DM basis) fed at 1% of BW daily. The Bermudagrass was conserved as hay without treatment (control), with NH(3) (30 g/kg of DM), or with a fibrolytic enzyme (16.5 g/t, air-dry basis) that was applied immediately after cutting (Ec), at baling (Eb), or at feeding. Chromic oxide was dosed to steers for 10 consecutive days, and fecal Cr concentrations from the last 5 d were used to estimate apparent total tract digestibility. In situ ruminal DM degradability was measured by incubating ground (4-mm) hay samples in duplicate in each of 2 ruminally cannulated cows having ad libitum access to Bermudagrass hay and 500 g/d of soybean meal. Unlike the enzyme treatment, ammoniation increased (P < 0.001) the CP concentration and reduced (P < 0.001) NDF, hemicellulose, and lignin concentrations of hay. Total DMI was greater (P < 0.05) for steers fed hays treated with Ec or NH(3) than for those fed control hays. All additive treatments increased (P < 0.05) DM digestibility, and NH(3), Ec, and Eb treatments also increased (P < 0.01) NDF digestibility. The initial and final BW, ADG, BCS, G:F, and hip height of the steers were not affected (P > 0.05) by treatment. The wash loss fractions in hays treated with Ec and Eb were lower than that in the control hay, but the potentially degradable fraction, total degradable fraction, and the effective degradability were increased (P < 0.01) by NH(3) treatment. Application at cutting was the most promising method of enzyme treatment, and this treatment was almost as effective as ammonia for enhancing forage quality.

Optimizing nitrogen utilization in growing steers fed forage diets supplemented with dried citrus pulp.

Our objectives were to compare the effects of sources of supplemental N on ruminal fermentation of dried citrus pulp (DCP) and performance of growing steers fed DCP and bahiagrass (Paspalum notatum) hay. In Exp. 1, fermentation of DCP alone was compared with that of isonitrogenous mixtures of DCP and solvent soybean meal (SBM), expeller soybean meal (SoyPLUS; SP), or urea (UR). Ground (1 mm) substrates were incubated in buffered rumen fluid for 24 h, and IVDMD and fermentation gas production kinetics and products were measured. Nitrogen supplementation increased (P < 0.10) ruminally fermentable fractions, IVDMD, pH, and concentrations of NH3 and total VFA, but reduced the rate of gas production (P < 0.10) and the lag phase (P < 0.01). Supplementation with UR vs. the soy-based supplements increased ruminally fermentable fractions (P < 0.05) and concentrations of total VFA (P < 0.10) and NH3 (P < 0.01), but these measures were similar (P > 0.10) between SBM and SP. In Exp. 2, 4 steers (254 kg) were fed bahiagrass hay plus DCP, or hay plus DCP supplemented with CP predominantly from UR, SBM, or SP in a 4 x 4 Latin square design, with four 21-d periods, each with 7 d for DMI and fecal output measurement. Nitrogen-supplemented diets were formulated to be isonitrogenous (11.9% CP), and all diets were formulated to be isocaloric (66% TDN). Intake and digestibility of DM, N, and ADF were improved (P < 0.05) by N supplementation. Compared with UR, the soy-based supplements led to greater (P < 0.05) DM and N intakes and apparent N and ADF digestibilities. Plasma glucose and urea concentrations increased (P < 0.10) with N supplementation and were greater (P < 0.01) for the soy-based supplements than for UR. Intake, digestibility, and plasma metabolite concentrations were similar (P > 0.1) for SBM and SP. In Exp. 3, 24 steers (261 kg) were individually fed bahiagrass hay plus DCP (control), or hay plus DCP supplemented with CP predominantly from UR or SBM. Over 56 d, DMI and ADG were greatest (P < 0.05) in steers fed SBM. Nitrogen supplementation increased (P < 0.05) DMI, ADG, and G:F. However, SBM supplementation produced greater (P < 0.05) DMI and ADG and similar (P > 0.05) G:F compared with UR supplementation. We conclude that supplemental N is important to optimize ruminal function and performance of growing steers fed forage diets supplemented with DCP. Diets with supplemental N mainly from SBM improved diet digestibility and animal performance beyond that achieved by UR.

Effects of dietary n-6:n-3 fatty acid ratio on feed intake, digestibility, and fatty acid profiles of the ruminal contents, liver, and muscle of growing lambs.

This study investigated the effect of modifying the n-6:n-3 fatty acid ratio (FAR) of diets using linseed, soybean, and cottonseed oils on apparent digestibility, ruminal fermentation characteristics, growth performance, key circulating hormones, and the fatty acid profile of ruminal digesta, liver, and fore-shank muscle of growing lambs fed a high concentrate diet. Forty individually housed Katadhin Dorper lambs (average of 20.0 kg of BW) were fed Bermudagrass hay in ad libitum amounts and concentrates at 3.7% of BW daily. The concentrate contained 68.9% corn, 23.8% soybean meal, 3.3% limestone, and 4.0% oil supplements (DM basis). The treatments consisted of dietary n-6:n-3 FAR of 2.3:1, 8.8:1, 12.8:1, and 15.6:1. After feeding for 35 d in metabolism crates, lambs were slaughtered 15 h after feeding, and samples of ruminal digesta, blood, liver, and foreshank tissue were collected. Increasing dietary n-6:n-3 FAR did not affect the intake of DM nor the apparent digestibility of DM, ether extract, NDF, or ADF, but did increase apparent digestibility of CP (linear, P < 0.05). Concentrations of ruminal butyrate increased linearly (P < 0.05) with increasing dietary n-6:n-3 FAR, whereas the valerate concentration decreased linearly (P < 0.001). Concentrations of plasma insulin and IGF-I were not affected by dietary n-6:n-3 FAR. Concentrations of C18:3n-3 increased linearly (P < 0.001), whereas that of C18:2n-6 decreased linearly (P < 0.001) in ruminal digesta with decreasing dietary n-6:n-3 FAR. Concentrations of transisomers of fatty acids in ruminal digesta did not change. Proportions of C18:0 in liver and foreshank muscle were unchanged by diet. The proportion of trans11 C18:1 and cis-9 trans11 CLA decreased (P < 0.05) in liver but increased (P < 0.05) in foreshank muscle as dietary n-6:n-3 FAR decreased. Proportions of all measured n-3 fatty acids were greater in liver when diets contained more C18:3n-3 from linseed oil. By decreasing the dietary n-6:n-3 FAR, the proportions of n-6 fatty acids in foreshank muscle decreased dramatically; specifically, C18:2n-6 decreased linearly (P < 0.001) from 28.0 to 16.5% and C20:4n-6 decreased linearly (P < 0.001) from 14.7 to 8.6%. Although feeding a diet that contained more n-3 fatty acids increased the n-3 fatty acid concentration of muscle, the ratio of PUFA to SFA was decreased.

Study on the inhibitory effects of Korean medicinal plants and their main compounds on the 1,1-diphenyl-2-picrylhydrazyl radical.

A 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-generating system was used to evaluate the antioxidant properties of Korean medicinal plants that have been used widely as folk medicines for several disorders, as well as compounds isolated from them. Among the Rosaceae, Rosa rugosa and Rosa davurica showed strong DPPH radical-scavenging activity. The most effective medicinal plant from families other than Rosaceae was Cedrela sinensis, followed in order by Nelumbo nucifera, Eucommia ulmoides, Zanthoxylum piperitum, Cudrania tricuspidata and Houttuynia cordata. These results serve as a good index of the free radical-scavenging activities of Korean medicinal plants. Furthermore, the polyphenols isolated from these plants, procyanidin B-3, (+)-catechin, gallic acid, methyl gallate, quercetin, quercetin-3-O-beta-D-glucoside, quercetin-3-O-beta-galactoside, quercetin-3-O-rutinose and kaempferol, exerted strong DPPH radical-scavenging activity. These results suggest that the Korean medicinal plants and the polyphenols isolated from them that exhibited effective radical-scavenging activity may be promising agents for scavenging free radicals and treating diseases associated with excess free radicals.

Synergistic effects of sildenafil on relaxation of rabbit and rat cavernosal smooth muscles when combined with various vasoactive agents.

OBJECTIVE: To evaluate which vasoactive agents have synergistic effects on the cavernosal smooth muscles of rabbits and rats when the agents are combined with sildenafil. MATERIALS AND METHODS: Relaxation responses of cavernosal smooth muscle to single agents (phentolamine, moxisylyte, sodium nitroprusside, forskolin, vasoactive intestinal peptide, VIP, papaverine and sildenafil) in the rabbit, and prostaglandin-E1 and sildenafil in the rat, and to combinations of each agent plus sildenafil, were assessed in vitro. The response to sildenafil of the rabbit strips with and without incubation with l-arginine (1 mmol/L) for 20 min was also evaluated. The effective concentrations for a half-maximal response of single agents and combination solutions were compared. RESULTS: All single agents induced concentration-dependent relaxation of the rabbit and rat cavernosal smooth muscles. There was significant synergism on rabbit cavernosal smooth muscle when the sildenafil was combined with forskolin, sodium nitroprusside, VIP or phentolamine. There was also significant synergism with sildenafil plus prostaglandin-E1 in rat cavernosal muscles. There were no synergistic effects of combinations of sildenafil plus moxisylyte, papaverine or l-arginine. CONCLUSIONS: These results suggest potentially effective combined therapies of sildenafil and intraurethral or intracavernosal prostaglandin-E1, intracavernosal forskolin or VIP, or oral phentolamine for patients with erectile dysfunction who have no success after monotherapy with these agents.

The contribution of epistasis to species differences in annual sunflowers.

The contribution of epistasis to 15 morphological traits differentiating two annual sunflowers (Helianthus annuus and H. debilis ssp. cucumerifolius) and to hybrid pollen sterility was estimated in a first generation backcross (BC1) mapping population. Analysis of digenic interactions among quantitative trait loci (QTLs) with significant main effects revealed significant interaction effects for six of the 15 morphological traits and for pollen sterility. Likewise, a genome-wide scan of all possible two-locus combinations detected additional significant interactions for three of the traits with significant epistasis in the original analysis: stem pigmentation, phyllary pubescence, and pollen viability. However, these were the only traits of the 16 examined in which detected interactions explained more than 5% of phenotypic variance. The implications of these findings for adaptive evolution and for the introgression of advantageous morphological QTLs across a natural hybrid zone between these taxa are discussed.

Molecular cloning and characterization of alternatively spliced transcripts of the turkey pituitary adenylate cyclase-activating polypeptide.

Pituitary adenylate cyclase-activating polypeptide (PACAP) increases the release of growth hormone (GH) and prolactin (PRL) in mammals. However, the evolutionary and functional relationships of PACAP, GH, and PRL are not clear. To understand how PACAP is regulated in the turkey, a turkey PACAP (tPACAP) cDNA has been cloned by the combination of reverse transcription-polymerase chain reaction and the rapid amplification of cDNA 5'- and 3'-ends. The deduced amino acid sequence of tPACAP-38 and turkey PACAP-related peptide (tPRP) displayed 87-97 and 52-63% similarity when compared to a variety of known PACAP-38 and PRP sequences, respectively. Two major transcripts (1.3 and 3.0 kb) of tPACAP were detected by Northern blot analysis. The highest levels of tPACAP mRNA were shown to be expressed in the hypothalamus, the cerebellum, and the cerebrum. In contrast, most of the other tissues tested expressed relatively low steady-state levels of tPACAP mRNA. Alternative splicing of tPACAP resulted in the expression of two different isoforms. The smaller form of tPACAP was expressed in the hypothalamus during early embryonic development and decreased significantly in later stages.

Characterization and cDNA cloning of two glycine- and histidine-rich antimicrobial peptides from the roots of shepherd's purse, Capsella bursa-pastoris.

Two novel antimicrobial peptides were isolated and characterized from the roots of shepherd's purse, Capsella bursa-pastoris. These antimicrobial peptides, named shepherin I and shepherin II, consist of 28 and 38 amino acids, respectively, and are glycine- and histidine-rich peptides. Shepherin I and shepherin II have 67.9% and 65.8% (mol/mol) glycine, respectively, and 28.6% and 21.1% (mol/mol) histidine, respectively. Both shepherins have a Gly-Gly-His motif. These antimicrobial peptides exhibit antimicrobial activity against Gram-negative bacteria and fungi. Circular dichroism spectra of shepherin I and shepherin II showed that shepherin I and shepherin II in 50% trifluoroethanol have 66.7% and 75% random coils, respectively, without any alpha-helices. cDNA sequence analysis revealed that shepherin I and shepherin II are produced from a single polypeptide, designated shep-GRP, consisting of 120 amino acids; shep-GRP has five distinct domains, an amino-terminal putative signal peptide, a shepherin I, a linker dipeptide, a shepherin II and a carboxy-terminal peptide. Southern blot analysis indicates that the gene encoding shepherins belongs to a low-complexity gene family. Northern blot analysis revealed that transcripts of shep-GRP are present in roots but not in leaves and stems.

Preparation of monoclonal antibody against ginsenoside Rf and its enzyme immunoassay.

A rapid and sensitive indirect competitive enzyme immunoassay method has been developed for quantitating ginsenoside Rf (Rf) in crude total Panax ginseng saponins and in rat plasma using high titer mouse monoclonal antibody (mAb) raised against a conjugate of Rf and bovine serum albumin (BSA). The isotype of mAb against Rf was IgG3 with a K chain. The presence of Rf inhibited the binding of the mouse anti-Rf mAb to a Rf-BSA solid phase coating antigen. The working range was 0.01-10 ng/assay and detection limits were 20 pg in various ginseng extract fractions or 34 pg in rat plasma per assay. The anti-Rf mAb cross-reacted with ginsenoside Rg2 by 57.5%, but not with other ginsenosides. However, this anti-Rf mAb did not cross-react with BSA or cellubiose, which is a carbohydrate component of Rf. Using this standard curve, we could measure the amount of Rf in ginseng total extract, ginseng total saponins, protopanaxadiol saponins, and propanaxatriol saponins. We could also measure the amount of Rf in rat plasma after the oral administration of Rf and found that Rf reached a maximum level in rat plasma after 16 h. These results indicate that the anti-Rf mAb could be useful for the quantitation of Rf in crude ginseng fractions and in body fluids.

Genetic architecture of species differences in annual sunflowers: implications for adaptive trait introgression.

Genetic architecture may profoundly influence the ability of adaptive traits to spread between species via introgressive hybridization. Here, we examine the genomic location of quantitative trait loci (QTL) associated with pollen sterility and morphological traits distinguishing two annual sunflowers, Helianthus annuus and H. debilis ssp. cucumerifolius. These species are of particular interest since they hybridize naturally, and the form of H. annuus in Texas (called ssp. texanus) is thought to have arisen through introgression. Analysis of 226 BC(1) progeny from a cross between H. annuus and H. debilis revealed 56 QTL for 15 morphological traits and 2 QTL for pollen sterility. Four morphological QTL are tightly linked (<10 cM) to one or more sterility factors and 7 are closely allied with underrepresented and presumably negatively selected chromosomal blocks. Although these 11 QTL seem unlikely to move between the species, no barrier to introgression was detected for the remaining 45 morphological QTL. In fact, due to widespread pleiotropy (or tight linkage), the introgression of just three small chromosomal blocks appears sufficient to largely recover the phenotype of ssp. texanus. Subsequent work will test for the occurrence and fitness consequences of the identified QTL in natural populations of ssp. texanus.

The antioxidative effect of ganhuangenin against lipid peroxidation.

The antioxidative effect of ganhuangenin (GHG), isolated from Scutellaria baicalensis Georgi, was examined by measuring its ability to suppress the formation of phospatidylcholine hydroperoxide (PCOOH). The results show that a pretreatment with GHG effectively suppressed PCOOH formation, which was initiated by the peroxyl-generating oxidant, AAPH (2,2'-azobis-2-aminopropane hydrochloride). The protective action of GHG against the formation of the PCOOH was observed in liver, lung, and kidney. When compared with other known antioxidants, we found the antioxidative potency of GHG to be greater than that of alpha-tocopherol. Our data strongly indicate that GHG is a powerful antioxidant against lipid peroxidation and is, therefore, responsible for this prophylactic effect.

Constitutive activation of extracellular signal-regulated kinase in human acute leukemias: combined role of activation of MEK, hyperexpression of extracellular signal-regulated kinase, and downregulation of a phosphatase, PAC1.

Extracellular signal-regulated kinase (ERK) is an important intermediate in signal transduction pathways that are initiated by many types of cell surface receptors. It is thought to play a pivotal role in integrating and transmitting transmembrane signals required for growth and differentiation. Constitutive activation of ERK in fibroblasts elicits oncogenic transformation, and recently, constitutive activation of ERK has been observed in some human malignancies, including acute leukemia. However, mechanisms underlying constitutive activation of ERK have not been well characterized. In this study, we examined the activation of ERK in 79 human acute leukemia samples and attempted to find factors contributing to constitutive ERK activation. First, we showed that ERK and MEK were constitutively activated in acute leukemias by in vitro kinase assay and immunoblot analysis. However, in only one half of the studied samples, the pattern of ERK activation was similar to that of MEK activation. Next, by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and immunoblot analysis, we showed hyperexpression of ERK in a majority of acute leukemias. In 17 of 26 cases (65.4%) analyzed by immunoblot, the pattern of ERK expression was similar to that of ERK activation. The fact of constitutive activation of ERK in acute leukemias suggested to us the possibility of an abnormal downregulation mechanism of ERK. Therefore, we examined PAC1, a specific ERK phosphatase predominantly expressed in hematopoietic tissue and known to be upregulated at the transcription level in response to ERK activation. Interestingly, in our study, PAC1 gene expression in acute leukemias showing constitutive ERK activation was significantly lower than that in unstimulated, normal bone marrow (BM) samples showing minimal or no ERK activation (P =.002). Also, a significant correlation was observed between PAC1 downregulation and phosphorylation of ERK in acute leukemias (P =.002). Finally, by further analysis of 26 cases, we showed that a complementary role of MEK activation, ERK hyperexpression, and PAC1 downregulation could contribute to determining the constitutive activation of ERK in acute leukemia. Our results suggest that ERK is constitutively activated in a majority of acute leukemias, and in addition to the activation of MEK, the hyperexpression of ERK and downregulation of PAC1 also contribute to constitutive ERK activation in acute leukemias.