Perilla Ketone Research Articles

Ligand characterization of CYP4B1 isoforms modified for high-level expression in Escherichia coli and HepG2 cells.

Human CYP4B1, a cytochrome P450 monooxygenase predominantly expressed in the lung, inefficiently metabolizes classical CYP4B1 substrates, such as the naturally occurring furan pro-toxin 4-ipomeanol (4-IPO). Highly active animal forms of the enzyme convert 4-IPO to reactive alkylating metabolite(s) that bind(s) to cellular macromolecules. By substitution of 13 amino acids, we restored the enzymatic activity of human CYP4B1 toward 4-IPO and this modified cDNA is potentially valuable as a suicide gene for adoptive T-cell therapies. In order to find novel pro-toxins, we tested numerous furan analogs in in vitro cell culture cytotoxicity assays by expressing the wild-type rabbit and variants of human CYP4B1 in human liver-derived HepG2 cells. To evaluate the CYP4B1 substrate specificities and furan analog catalysis, we optimized the N-terminal sequence of the CYP4B1 variants by modification/truncation and established their heterologous expression in Escherichia coli (yielding 70 and 800 nmol·l-1 of recombinant human and rabbit enzyme, respectively). Finally, spectral binding affinities and oxidative metabolism of the furan analogs by the purified recombinant CYP4B1 variants were analyzed: the naturally occurring perilla ketone was found to be the tightest binder to CYP4B1, but also the analog that was most extensively metabolized by oxidative processes to numerous non-reactive reaction products.

Establishing Modified Human CYP4B1 As a Novel Suicide Gene System for Adoptive T-Cell Therapies

Retargeting autologous T-cells to recognize tumor-associated antigens by genetic modifications with viral vectors that express either enhanced T-cell receptors (TCRs) or chimeric antigen receptors (CARs) has shown successful activity against B-cells malignancies. Equipping the transduced T-cell with a suicide gene can prevent potential safety problems as on-target off-target toxicity, off-target reactivity and cytokine-release syndrome (Casucci et al., 2015). The concept of a suicide gene has already been utilized to prevent a Graft-versus-host-disease (GvHD) following donor lymphocyte infusion (DLI). The first suicide gene system used in clinical phase I/II studies was the human Herpes simplex virus type 1 thymidine kinase with its prodrug ganciclovir that was used to control Graft-versus-host-disease following donor lymphocyte infusion (DLI) for leukemia relapse after allogeneic stem cell transplantation. More recent examples for suicide gene systems are icasp9, an inducible apoptosis switch with a synthetic dimerizer protein as prodrug, and CD20 with rituximab as a prodrug. We recently published a novel potential human suicide gene system by rendering the inactive P450 cytochrome CYP4B1 with 13 amino acid changes into the highly active CYP4B1P+12 in combination with 4-ipomeanol (4-IPO) (Wiek et al., 2015). 4-IPO is furan produced by sweet potatoes infected with the common mold Fusarium javanicum and has been identified as the major lung toxin causing lung edema in livestock (Wilson et al., 1970). A second natural occurring substance causing lung edema in horses is the major component of the Perilla frutenscens fruit oil called perilla ketone (PK) (Wilson et al., 1977). Importantly, the only difference between PK and 4-IPO is a methyl group at C5 in PK, where 4-IPO has a hydroxyl group. Therefore in this work, we tested the hypothesis whether PK could function as a potential new prodrug for the CYP4B1P+12 suicide gene.Initially we compared the cytotoxicity caused by PK and 4-IPO in CYP4B1P+12 transduced primary human T-cells from healthy donors. For these experiments, we used third generation lentiviral vectors with T2A sites to express the suicide gene in combination with a truncated version of the low affinity nerve growth factor receptor (DNGFR) as a selection marker for Miltenyi MACS microbeads. Through the selection process, we were able to enrich the suicide gene expressing T-cell population from 61±6.0% to ≥95±1.8%.After selection, we challenged the CYP4B1P+12 expressing T-cells with increasing concentrations (2.9-90 µM) of both substrates and analyzed the survival of the cells. After 24h, 65.8±5.26% of CYP4B1P+12 positive T-cells incubated with 2.9 µM 4-IPO were still alive, while incubation with 90 µM lead to only 17.5±1.45% living T-cells. In contrast, incubation with 2.9 µM PK resulted in only 16.02±1.08% living cells, while 90 µM PK induced cell death in 94.2±0.79% of cells. Next, we demonstrated that a lower concentration, 0.9 µM, already led to only 13.2±0.46% alive cells after 24h. Monitoring apoptosis in the transduced T-cells by flow cytometry demonstrated that incubation with 90 µM 4-IPO for 4h decreased the rate of transduced T-cells to only 87.4±2.89%, while 10h and 24h incubation resulted in 20.4±4.9% and 5.8±0.93% living annexinV-negative cells. In contrast, 2h incubation with only 9 µM PK led to 88.7±2.07%, 4h to 60.2±5.47% and 10h to only 5.5±1.85% living annexinV-negative T-cells. Incubation of the T-cells with 90 µM did not change these numbers. Hence, we were able show that PK is not only more potent as 4-IPO, it also proves to have a faster kinetic in inducing apoptosis.After these promising in vitro results of using PK as a novel more potent prodrug for the CYP4B1P+12 human suicide gene, we performed dose escalation studies of PK in mice with targeted disruption of the Cyp4b1 gene. Using up to 25 mg PK/kg mouse daily up for 3d did not induce any clinical symptoms in the Cyp4b1-/- animals, while Cyp4b1+/+ animals needed to be sacrificed 4h and 7h after a single i.p. injection of 10 mg/kg or 5 mg/kg PK, respectively, due to severe respiratory distress. Results of in vivo studies analyzing the elimination of CYP4B1P+12 positive syngenic T-cells in Cyp4b1-/- mice after injection with either 4-IPO or PK will be finalized and presented at the meeting. DisclosuresNo relevant conflicts of interest to declare.

Chemical Composition and Antibacterial Activity of the Essential Oils of Laggera crispata (Vahl) Hepper & Wood, Cyclospermum leptophyllum (Pers.) Eichler and Perilla frutescens (L.) Britton

Laggera crispata (Vahl) Hepper & Wood (Asteraceae), Cyclospermum leptophyllum (Pers.) Eichler (Apiaceae) and Perilla frutescens (L.) Britton (Lamiaceae) are under-explored aromatic plants of western-Himalaya. The hydrodistilled essential oils of L. crispata, C. leptophyllum and P. frutescens were analyzed using gas chromatography-flame ionization detector (GC-FID) and GC-mass spectrometry (GC-MS) and tested against nine pathogenic bacterial strains using disc diffusion assay. Major constituents of L. crispata essential oil were 2,5-dimethoxy-p-cymene (43.2 %), 10-epi-γ-eudesmol (19.7 %), eudesm-7(11)-en-4-ol (4.8 %), α-humulene (3.8 %), 7-epi-α-eudesmol (3.6 %) and (E)-caryophyllene (3.5 %). Major constituents of C. leptophyllum oil were 2,5-dimethoxy-p-cymene (46.8 %), thymol methyl ether (14.6 %), p-cymene (13.9 %), γ-terpinene (8.9 %) and carvacrol methyl ether (7.5 %). However, the essential oil of P. frutescens contained perilla ketone (48.6 %) and isoegomaketone (32.1 %) as main constituents. The essential oil of L. crispata exhibited good activity against Streptococcus mutans and moderate activities against Staphylococcus aureus and Klebsiella pneumoniae. The essential oil of C. leptophyllum showed moderate activity against Gram-positive bacteria, namely, Staphylococcus aureus (MTCC2940), Staphylococcus aureus (MTCC96) and Streptococcus mutans, where as the P. frutescens essential oil showed good activity against Streptococcus mutans and Bacillus subtilis. In general, the examined essential oils exhibited moderate to good activity against most of the tested Gram-positive bacteria, except L. crispata, which also showed moderate activity against Gram-negative strain, Klebsiella pneumoniae.

Anti-Bacillus and Anti-Shigella Activities of the Essential Oil from Perilla fruescens var. japonica Hara

To develop new effective and safe natural antibiotics against antibiotic-resistant food-borne pathogenic bacteria, the essential oil was extracted from the leaves of Perilla frutescens var. japonica Hara. Its composition was analyzed by GC-MS and its activities were tested against antibiotic-susceptible and resistant strains of some food-borne bacteria that can cause severe clinical symptoms in humans and animals. In addition, we determined the capacity of this oil to modulate the resistance of Bacillus and Shigella to antibiotics by checkerboard-titer tests on the basis of their MICs (minimum inhibitory concentrations). The essential oil fraction of P. frutescens var. japonica and its main component, perilla ketone, showed significant inhibitory activity against most of the tested strains of antibiotic-susceptible and resistant bacteria, resulting in MICs ranging from 0.25 to >2.00 mg/ml. The Perilla oils combined with norfloxacin or trimethoprim/ sulfamethoxazole showed synergistic or additive effects against Bacillus and Shigella species with FICIs ranging from 0.09 to 1.00 in the checkerboard-titer tests. In addition, the Perilla essential oil fraction and perilla ketone exhibited antioxidant activity with IC50s of 55.45 mg/ml and 71.13 mg/ml, respectively, in DPPH scavenging evaluation tests. Therefore, the essential oil fraction of P. frutescens var. japonica and its main component could be useful agents in the treatment of Bacillus and Shigella infections and as safe additives in food materials to prevent contaminations by these bacteria, especially in the face of the rapid increase of antibiotic-resistant strains, which can cause severe symptoms in humans. In addition, the significant antioxidant activity of this oil fraction or perilla ketone could provide benefits when applied as food preservatives.

Inhibition of Proinflammatory Cytokine Generation in Lung Inflammation by the Leaves of Perilla frutescens and Its Constituents

This study was designed to find some potential natural products and/or constituents inhibiting proinflammatory cytokine generation in lung inflammation, since cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) are pivotal for provoking airway inflammation. In our preliminary screening procedure, the 70% ethanol extract of the leaves of Perilla frutescens (PFE) was found to clearly inhibit TNF-α production in the lung at 100 mg/kg, after intranasal lipopolysaccharide treatment of mice. Based on this result, ten constituents including phenylpropanoids (allyltetramethoxybenzene, caffeic acid, dillapiole, elemicin, myristicin, nothoapiole, rosmarinic acid methyl ester, rosmarinic acid) and monoterpenes (perilla aldehyde and perilla ketone) were successfully isolated from the extract. Among them, elemicin and myristicin were found for the first time to concentration-dependently inhibit IL-1β-treated IL-6 production from lung alveolar epithelial cells (A549) at concentrations of 10-100 μM. These findings suggest that the phenylpropanoids including elemicin and myristicin have the potential to be new inhibitory agents against lung inflammation and they may contribute, at least in part, to the inhibitory activity of PFE on the lung inflammatory response.

Volatile oil composition of Indian Perilla [Perilla frutescens (L.) Britton] collected at different phenophases

Volatile oils of the Indian Perilla [Perilla frutescens (L.) Britton], isolated from different phenophases, were analyzed by gas chromatography/flame ionization detector (GC/FID) and GC–mass spectrometry (GC/MS). Essential oil yield was varied from 0.26% to 0.35% during different phenophases of P. frutescens. However, leaves and inflorescence possessed 0.50% and 0.25% essential oil, respectively. Major constituents of the oil were perilla ketone (39.5–59.3%), isoegomaketone (9.4–45.7%), (E)-caryophyllene (0.1–13.4%), and caryophyllene oxide (0.4–3.8%). Perilla ketone was found to be higher at seed maturity stage (59.3%), while the amount of isoegomaketone was recorded higher during vegetative stages (39.8–43.6%). Further, perilla ketone and (E)-caryophyllene were found to be higher in inflorescence, while isoegomaketone was higher in leaves. The investigated P. frutescens chemotype (perilla ketone/isoegomaketone) is new to India.

Compositional variability in volatiles from different plant organs of Perilla frutescens L. cultivated in Uttarakhand (India)

Abstract Background This investigation aims to determine the variation in essential oil compositions in plant parts (whole plant, leaves, spikes and husk) of Perilla (Perilla frutescens L.) at 3 different sowing times and also to ensure the suitability of this crop in Doon valley climatic conditions for commercial cultivation. Methods During the course of study, seeds were sown at 25 days intervals in the month of May, June and July with 60 × 30 cm spacing. The essential oils from all the samples were hydro-distilled using a Clevenger-type apparatus and analyzed by GC/MS. Results and discussion In all the oils, perilla ketone (43.49–90.28%) and 1-methyl-2-methylene trans-decalin (4.49–36.11%) were found to be the most abundant compounds. Conclusion On the basis of comparative composition, D1 stage (seeds sown on 20th May) showed better results as compared with D2 (seeds sown on 15th June) and D3 (seeds sown on 10th July).

Essential oil composition and antimicrobial activity of Lobelia pyramidalis Wall.

The essential oil of Lobelia pyramidalis was analyzed by GC and GC-MS. A total of 21 constituents comprising 77.88 % of the total oil were identified. Perilla ketone constituted 25.61 % of the oil followed by camphorquinone (12.16 %), dibutyl phthalate (10.66 %) and allyl nonanoate (8.47 %). The antimicrobial activity of the oil was evaluated using the disc diffusion method and the microdilution technique. The results showed that the oil exhibited moderate antimicrobial activity.

Comparison of HS-SPME with hydrodistillation and SFE for the analysis of the volatile compounds of Zisu and Baisu, two varietal species of Perilla frutescens of Chinese origin

Content and composition of the volatiles of Baisu and Zisu, two varieties of Perilla frutescens, obtained by SFE, HS-SPME, and hydrodistillation, were compared. A total of 64 compounds were identified by GC/MS. Perillaldehyde (21.9–58.1%) and perilla ketone (54.5–83.5%) were the main volatile components of Zisu and Baisu, respectively. They might be partly responsible for the aroma and taste of perilla. The abundance of perilla ketone indicates that it is not safe to use Baisu as vegetable, and Baisu should not be used as substitute for Zisu. SFE extracted oil with high yield, and extracted some high molecular-weight compounds that did not contribute to the aroma. The HS-SPME was simple and sensitive for the analysis of volatile compounds of fresh perilla. The results showed that the drying process influenced the volatile components of perilla.

Characteristic Aroma-Active Compounds of Korean Perilla (Perilla frutescens Britton) Leaf

Aroma-active compounds from Korean perilla (Perilla frutescens Britton) leaf were extracted by solvent-assisted flavor evaporation (SAFE), liquid-liquid continuous extraction (LLCE), and hydrodistillation (HD) and analyzed by gas chromatography-mass spectrometry (GC-MS) and gas chromatography-olfactometry (GC-O). Thirty-three volatile compounds were identified by GC-MS. 1-(3-Furyl)-4-methyl-1-pentanone (perilla ketone) was found to be the most abundant volatile compound, followed in order by (Z)-3-hexenol and 1-octen-3-ol. Perilla ketone comprised 81% (93 ppm), 84% (120 ppm), and 95% (490 ppm) of the volatile compounds obtained from SAFE, LLCE, and HD, respectively. Thirteen aroma-active compounds were detected by GC-O. Perilla ketone, 1-(3-furyl)-4-methyl-3-penten-1-one (egoma ketone), and 1-(3-furyl)-4-methyl-2-penten-1-one (isoegoma ketone) were considered to be the characteristic aroma-active compounds of Korean perilla leaf. Perilla ketone, (Z)-3-hexenal (green), egoma ketone, and isoegoma ketone were the most intense aroma-active compounds in Korean perilla leaf. Other relatively intense odorants included (Z)-3-hexenol (green), (E)-2-hexenal (green), benzaldehyde (almond), 1-octen-3-one (metallic), 1-octen-3-ol (mushroom), phenylacetaldehyde (honeysuckle), linalool (lemon), and beta-caryophyllene (woody).

Determination of toxic perilla ketone, secondary plant metabolites and antioxidative capacity in five Perilla frutescens L. varieties

Perilla frutescens L. is a traditional Asian crop with multiple uses. Several varieties exist but only little data is available about the content of the toxic perilla ketone and secondary plant metabolites of those genotypes. To estimate the nutritional value of this new vegetable more information about those components is necessary. We investigated five genotypes of P. frutescens L. to determine their content of PK, phenolics, carotenoids and AC. AC was examined using ABTS-decolorization assay and lipid peroxidation assay. Carotenoids were identified and quantified by HPLC analysis, phenolics were quantified by means of Folin–Ciocalteu and PK was identified by GC/MS. Two genotypes were found to contain PK, a potent lung toxin, and are therefore not suitable for consumption. The phenolic content and corresponding antioxidative capacity of all genotypes is considerably high compared to other vegetables. All genotypes moreover contain notably high concentrations of carotenoids with contents up to fivefold higher than in other carotenoid rich vegetables. The results indicate that there are several genotypes which are not suitable for consumption due to their content of PK. However PK free genotypes are rich sources of natural antioxidants, and may therefore be considered as a novel vegetable with health promoting properties.

Essential oil variations in different Perilla L. accessions: chemotaxonomic implications

In 2006, 26 accessions (mainly from Sichuan province, China) in one species and two varietal species among the genus Perilla L. were collected, and the essential oils (EOs) of their fresh leaves during the flowering period were analyzed by the GC-MS method in 2007. In total, 49 components could be identified. The results showed that eight chemotypes could be identified as follows: PK (perillaketone), DLP (d-limonene and piperitone), PT (piperitenone), MS (myristicin), AL (apiol), EM (elemicin), DEK (dehydroelsholtzia ketone) and PA (perilla aldehyde). Of them, chemotype DLP, AL and DEK were reported for the first time; DLP and AL were found only in the accessions from Sichuan province, China. In our study, the EOs of P06-1Z and P06-28 were investigated at several stages during growing. Taking into account the variability of the two accessions during the year, the seasonal influence on the main constituents could not change the status of the different chemotypes distinguished or enhance the veracity of the differentiation of eight chemotypes. The results also suggested that it may not always be possible to establish a correlation between the traditional morphological taxonomy and chemotypes.

밀양지역 들깨품종별 잎의 아미노산 조성 및 향기성분 비교

우리나라 들깻잎의 주산지인 밀양지역에서 재배하고 있는 4종의 품질 특성을 조사하고자 아미노산과 휘발성 향 성분을 분석하여 비교하였다. 단백질구성아미노산에서 주요 아미노산은 aspartic acid, glutamic acid, glycine, alanine, arginine, 및 threonine 등 이었고, 그 외에 lysine, leucine, isoleucine 등도 다소 검출되었으나, tyrosine과 함황아미노산인 cysteine과 methionine의 함량이 가장 적 게 나타났으며, 유리 아미노산은 4종 모두에서 약34종이 검출되었다. 그리고 유리아미노산의 조성과 단백질구성아미노산의 조성에서 품종간의 차이는 거의 없는 것으로 나타났다. 들깻잎의 휘발성 향기성분을 GC-MSD로 분석한 결과, 잎들깨1호는 52종, 엽실들깨는 48종, 그리고 밀양9호와 YCPL은 47종이 분리되어 검출되었다. 깻잎의 휘발성 화합물중에서 가장 많은 함량으로 검출된 perilla ketone은 엽실들깨 <TEX>$551.42\;{\mu}g/g$</TEX>, 잎들깨1호 <TEX>$301.59\;{\mu}g/g$</TEX>, YCPL <TEX>$187.00\;{\mu}g/g$</TEX>, 그리고 밀양9호는 <TEX>$145.75\;{\mu}g/g$</TEX>이었으며, 그 외 3-hexen-1-ol, trans-2-hexenal, <TEX>$\beta-caryophyllene$</TEX> 그리고 <TEX>$\alpha-farnesene$</TEX>등이 검출되었으나 이 화합물들의 함량은 <TEX>$20\;{\mu}g/g$</TEX>을 초과하지 않았다. The present investigation was carried out to determine the qualitative difference between 4 varieties of perilla leaves cultivated Miryang area, particularly focusing on the amino acid composition and flavor compounds. Aspartic acid, glutamic acid, glycine, alanine, arginine, and threonine were the major amino acids, and tyrosine and cysteine and methionine containing sulfur were detected as little amount in protein of all perilla leaves. All perilla leaves contained about 34 kinds of free amino acids, and the major free amino acids were same as the protein bound amino acids. However, no significant difference among 4 varieties of perilla leaves was observed in the profiles of protein bound and free amino acids composition. Flavor compounds analyzed by GC-MSD following to extraction of flavor by SDE (Simultaneous Steam Distillation-Extraction) were detected as 51 kinds from 'Leafy perilla 1', 47 kinds from 'Yupsil perilla', 46 kinds from 'Miryang 9' and 'YCPL. The major volatile compound was perilla ketone, its concentration was <TEX>$145.75\;{\mu}g/g$</TEX> in 'Miryang 9', <TEX>$187.00\;{\mu}g/g$</TEX> in 'YCPL', <TEX>$301.59\;{\mu}g/g$</TEX> in 'Leafy perilla l' and <TEX>$551.42\;{\mu}g/g$</TEX> in 'Yupsil perilla', but the other flavor compounds, 3-hexen-l-ol, trans-2-hexenal, <TEX>$\beta-caryophyllene,\;\alpha-farnesene$</TEX> and etc, were less than <TEX>$20\;{\mu}g/g$</TEX>.

Neutral and DEAE dextrans as tracers for assessing lung microvascular barrier permeability and integrity.

Steady-state lymph-to-plasma concentration ratios (L/Ps) of neutral dextrans, cationic DEAE dextrans, and endogenous proteins were determined under normal and increased permeability conditions in six unanesthetized yearling sheep prepared with chronic lung lymph fistulas. Fluorescent dextrans with radii ranging from 1 to 30 nm were intravenously infused, and after 24 h, perilla ketone (PK) was given to alter permeability while the dextran infusion was maintained. Plasma and lymph samples were collected before and after PK administration and analyzed for dextran and protein concentrations after high-performance liquid chromatography size separation. Under both baseline and increased permeability conditions, DEAE dextrans had higher L/Ps than neutral dextrans of similar size but lower L/Ps than proteins of similar size. Comparison of L/Ps before and after PK revealed that the percentage change in permeability for neutral and DEAE dextrans was significantly larger than that for proteins. These results suggest that 1) the pulmonary microvascular barrier behaves as a net negative barrier, 2) some transport mechanisms for proteins and dextrans are different, and 3) neutral and cationic dextrans are more sensitive markers than proteins of the same size for assessing changes in pulmonary capillary permeability.

Correlation between Increased Airway Responsiveness and Severity of Pulmonary Edema

To determine whether the severity of the pulmonary edema in sheep models of cardiogenic and non-cardiogenic pulmonary edema correlate with concomitant alterations in airway responsiveness using three separate measures of pulmonary edema: post-mortem wet-to-dry lung weight ratio (W/D), chest radiograph (CXR) scores, and small airway wall area. Cardiogenic pulmonary edema was induced by increasing left atrial pressure (↑mPLA) and non-cardiogenic pulmonary edema was induced by intravenous administration of Perilla ketone (PK). There was a significant negative correlation between changes in airway responsiveness and changes in CXR grade (r=−0.749, P<0.05), W/D (r=−0.662, P<0.05), airway wall areas (r=0.784, P<0.05) after increases in both PLA and PK. Chest radiograph score, W/D, and airway wall area correlated with each other (CXR score and W/D r=0.657, P<0.05; CXR score and airway wall area r=0.678, P<0.05; airway wall area and W/D r=0.704, P<0.05). We speculate that the increased airway responsiveness observed during pulmonary edema may result from the mechanical effects of edema formation within the airways.

The effects of roasting temperatures on the formation of headspace volatile compounds in perilla seed oil

AbstractVolatile compounds of perilla seed oils roasted at different temperatures (150–190°C) were analyzed by dynamic headspace gas chromatography‐mass spectrometry. The headspace volatiles in roasted perilla seed oils (RPSO) were composed of thermally produced flavors and compounds originating from the raw perilla seeds. The roasting temperatures significantly affected the production of thermal reaction flavors. Oils from parilla seeds roasted below 170°C had relatively high concentrations of aldehydes, whereas pyrazines and furans were the predominant volatiles above 170°C. In all of the RPSO, the contents of both perilla aldehyde and perilla ketone remained almost constant and might be used to discriminate perilla seed oils from other roasted vegetable seed oils.

Effect of Pulmonary Edema on Tracheal Diameter

Background: Though it is well known that cardiogenic and noncardiogenic pulmonary edema can cause changes in lung mechanics, actual alterations in tracheal diameter have not been described. Objective: To evaluate the effects of pulmonary edema induced by increased left atrial pressure (cardiogenic) and Perilla ketone (PK; noncardiogenic) on tracheal diameter in chronically instrumented awake sheep. Methods: We investigated the effects of two mechanistically distinct types of pulmonary edema on tracheal diameter in chronically instrumented awake sheep. Cardiogenic pulmonary edema (analogous to congestive heart failure in humans) was induced by increasing left atrial pressure (↑P_LA) by inflating the balloon on a Foley catheter positioned in the mitral valve annulus to cause partial obstruction to flow across the valve (n = 18). Noncardiogenic pulmonary edema (increased pulmonary microvascular permeability pulmonary edema analogous to the acute respiratory distress syndrome in humans) was produced by the intravenous administration of PK (n = 11). Lateral chest radiographs (CXRs) were scored by a standardized 5-point scoring system for the severity of pulmonary edema, and tracheal diameter was measured at a fixed location in the carina. Three radiologists, blinded to sheep identification number and experimental protocol, evaluated the radiographs independently at different points in time for edema severity and tracheal diameter. The sheep were sacrificed immediately after the final CXR, and wet/dry lung weight ratio (W/D ratio) was determined. Results: Both ↑P_LA and PK were associated with statistically significant tracheal narrowing (↑P_LA: 20.3 ± 0.6 to 15.1 ± 0.9 mm; PK: 20.2 ± 0.6 to 14.1 ± 1.4 mm). Tracheal narrowing correlated with the severity of the pulmonary edema determined radiographically (↑P_LA: r = –0.69, p < 0.01; PK: r = –0.62, p < 0.01) and by W/D ratio (↑P_LA: r = –0.64, p < 0.05; PK: r = –0.54, p < 0.05). Conclusions: We conclude that tracheal narrowing occurs in sheep models of both cardiogenic and noncardiogenic pulmonary edema and that the degree of narrowing correlates with the severity of the edema.

Effect of cardiogenic and noncardiogenic pulmonary edema on histamine responsiveness in sheep.

We compared the effects of cardiogenic pulmonary edema, brief pulmonary vascular congestion without frank edema, and noncardiogenic pulmonary edema on responsiveness to inhaled histamine in chronically instrumented awake sheep. Histamine responsiveness was measured before and after 1) cardiogenic pulmonary edema induced by raising left atrial pressure to 35 cmH2O (Pla) for 3.5 h by partial obstruction of flow across the mitral valve, 2) brief cardiogenic congestion via Pla for 0.5 h, 3) noncardiogenic pulmonary edema induced by 25 mg/kg intravenous perilla ketone (PK), and 4) 3.5 h of monitoring without Pla or PK (controls). Treatment for 3.5 h with Pla (n = 9) and PK (n = 11) each significantly lessened the histamine dose required to cause a fall to 65% of baseline dynamic lung compliance (ED65Cdyn), i.e., increased responsiveness. Sheep treated for 0.5 h with Pla (n = 7) and controls (n = 5) showed no significant change in ED65Cdyn. Intravenous atropine (0.1 mg/kg) before the second histamine challenge altered neither the reduction of ED65Cdyn in Pla (n = 8) and PK (n = 9) sheep nor the ED65Cdyn level of controls (n = 9). These data imply that the local effects of edema, rather than bronchial vascular hemodynamics, cholinergic reflexes, and permeability changes, are germane to lung hyperresponsiveness during pulmonary edema in sheep.

Reamed intramedullary femoral nailing after induction of an "ARDS-like" state in sheep: effect on clinically applicable markers of pulmonary function.

At present, the optimal treatment for appropriately resuscitated, multiply injured patients includes fixation of long bone fractures within twenty-four hours of injury. This management approach has been shown to decrease the incidence of pulmonary complications, multiple organ failure, and death. Some investigators have hypothesized that acute reamed intramedullary nailing of the femur (RIMNF) may result in pulmonary dysfunction as a result of the pulmonary fat embolization generated during this procedure. Patients with concomitant thoracic trauma may be at particular risk for this potentially severe complication. In an attempt to determine whether RIMNF can be safely carried out regardless of the severity of a pulmonary injury, we monitored the pulmonary effects of RIMNF in sheep in which an acute respiratory disorder (ARDS)-like state had been induced. Our hypothesis was that, if the pulmonary fat embolization that occurs as a result of RIMNF has a clinically significant effect, it would be detectable in an animal model in which a severe lung injury had been induced prior to the start of RIMNF. This was an acute experimental procedure performed on yearling sheep. Reamed intramedullary nailing of the femur was performed in two groups of instrumented sheep. The first group had no pulmonary injuries. The second group had an ARDS-like state induced by intravenous infusion of perilla ketone prior to RIMNF. Perilla ketone increases pulmonary microvascular permeability without changing filling pressures and is used to induce a model of human ARDS. Hemodynamic and oximetric parameters were measured or calculated, as was pulmonary dynamic compliance during the experiment. Infusion of perilla ketone caused a significant pulmonary injury. RIMNF caused no additional significant effect on intrapulmonary shunt, mixed venous oxygen saturation, or dynamic compliance, which are clinically used to assess the severity of pulmonary dysfunction in injured patients. The fat embolization that occurs during RIMNF in an appropriately resuscitated sheep has no clinically significant effect on pulmonary function, even in the setting of a severe pulmonary dysfunction.

Genetic analysis of essential oil variants in Perilla frutescens.

A new chemotype (C type) of Perilla frutescens (Labiatae) that accumulates trans-citral as a main component of the essential oil in the leaf was crossed with five other chemotypes containing perillaldehyde (PA), elsholtziaketone (EK), perillaketone (PK), perillene (PL), and phenylpropanoid (PP) as their respective major components for comparison of genetic differences. The analyses of F1 and F2 progenies showed that trans-citral is accumulated when its metabolism is blocked in the simultaneous absence of a dominant gene N, which is involved in the conversion of trans-citral into naginataketone via cis-citral, and two polymeric genes Fr1 and Fr2, which are involved in the conversion of trans-citral into perillene. On the basis of new data obtained from various intercrosses involving the C type as one of the parents, the genotypes of different chemotypes as well as the sites of action of several genes controlling reaction steps in the biosynthesis of monoterpenes in Perilla have been revised.