Environmentally-Friendly Pesticidal Activities of Callicarpa and Karomia Essential Oils from Vietnam and Their Microemulsions

There is an ongoing interest to identify alternative pesticidal agents to avoid the chronic problems associated with synthetic pesticides. Essential oils have shown promise as botanical pest control agents. In the present study, the essential oils of four members of the Lamiaceae (Callicarpa candicans, C. erioclona, C. macrophylla, and Karomia fragrans; Vietnamese names: Nàng nàng, Tu châu lông mem, Tu châu lá to and Cà diện, respectively), obtained from wild populations in Vietnam, have been obtained by hydrodistillation and analyzed by gas chromatography-mass spectrometry. The essential oils were formulated into microemulsions and the essential oils and their microemulsions were screened for mosquito larvicidal activity against Aedes aegypti, Aedes albopictus, Culex quinquefasciatus, and for molluscicidal activity against Pomacea canaliculata. Atractylone and (E)-caryophyllene dominated the volatiles of C. candicans (CCEO) and C. erioclona (CEEO), while the major component in C. macrophylla (CMEO) and K. fragrans (KFEO) was (E)-caryophyllene. The essential oils and microemulsions of both C. candicans and C. erioclona exhibited excellent larvicidal activity against all three mosquito species (Ae. aegypti, Ae. albopictus, and Cx. quinquefasciatus) with LC₅₀ values <10 μg/mL. Additionally, the larvicidal activity of the microemulsions were significantly improved compared with their free essential oils, especially for C. candicans and C. erioclona. All four essential oils and their microemulsions showed excellent molluscicidal activity with LC₅₀ <10 μg/mL. In most cases, the essential oils and microemulsions showed greater pesticidal activity against target organisms than the non-target freshwater fish, Oreochromis niloticus. The in silico studies on physicochemical and ADMET properties of the major components in the studied essential oils were also investigated and most of the compounds possessed a favorable ADMET profile. Computational modeling studies of the studied compounds demonstrated a favorable binding interaction with the mosquito odorant-binding protein target and support atractylone, β-selinene, and caryophyllene oxide as potential inhibitors. Based on the observed pesticidal activities of the essential oils and their microemulsions, the Callicarpa species and K. fragrans should be considered for potential cultivation and further exploration as botanical pesticidal agents.     

Synthesis of [17,18-3H] trans-13-azaprostanoic acid. A labeled probe for the PGH2/TXA2 receptor

Because of its highly unstable nature, TXA2, produced by platelet metabolism of arachidonic acid, does not lend itself to use as a receptor probe for its own receptor. As such, the stable TXA2/PGH2 antagonist, trans-13-azaprostanoic acid (trans-13-APA, 12b), was prepared as the [17, 18 3H] derivative [( 3H] trans-13-APA, 12c) to study this receptor and to better evaluate the mechanism of action of these azaprostanoids. Tritiated trans-13-APA, 12c, was prepared in nearly theoretical specific activity (57 Ci/mmole) from (17Z)-trans-13-azaprost-17-enoic acid (11b) by catalytic tritiation. The unsaturated 11b was prepared by condensation of cis-7-amino-3-heptene (8) with 2-(6-carboxyhexyl) cyclopentanone (9), NaBH4 reduction, chromatography, and hydrolysis of the trans isomer so isolated. The olefins 11a and b were also of biochemical interest because of the unsaturation in the lower side chain. The presence of similar unsaturation in PGH3(4) and TXA3 (3) renders these prostaglandins inactive as proaggregatory agents. Evaluation of the antiaggregatory activity of 11a and b indicated it to be about the same potency in inhibiting human platelet aggregation as the parent cis and trans-13-APAs, suggesting that introduction of a double bond at the 17 position in platelet prostaglandin antagonists is unlikely to result in enhanced antiplatelet activity.     

Spin-label studies of membrane-associated denatured hemoglobin in normal and sickle cells

A maleimide spin label (N-(1-oxyl-2,2,5,5-tetramethylpyrrolidinyl)-maleimide) was reacted with oxyhemoglobin-free cell stromata of normal and sickle cells. The EPR spectrum of spin-labeled red cell membranes showed that the spin labels are attached to at least two different binding sites. There was a major signal, A, which characterized a strongly immobilized environment and a minor signal, B, which characterized a weakly immobilized environment. Quantitative EPR measurements using equal amounts of Hb AA and Hb SS red blood cells demonstrated that Hb SS red cell membranes had an approximately four times higher EPR signal intensity than Hb AA red cell membranes ((7.98 ± 1.14) · 10⁵ and (2.2 ± 1.2) · 10⁵ spin labels/cell, respectively). Moreover, the ratio of signal intensities A and B are different in these cells. Comparative spectrophotometric studies of membrane-associated denatured hemoglobins of Hb AA and Hb SS red cell membranes suggested that the EPR signal A is derived from spin labels attached to membrane-associated denatured hemoglobin, while signal B is mainly from spin labels attached to membrane-associated denatured hemoglobin, while signal B is mainly from spin labels attached to membranes. The combination of EPR spectrum of Hb AA membranes pretreated with N-ethyl-maleimide and that of spin-labeled precipitated hemoglobin further strengthened this conclusion.     

Vascularization of plexus myentericus (Auerbach) in the large intestine of the cat

1. Coincidental preparation of the intramuscular vascular bed and the plexus myentericus (Auerbach) of the cat's large intestine by India-ink method and silverimpregnation allowed to demonstrate independent vascularisation of ganglia and nerve-branches of the plexus Auerbach. 2. Each ganglion is surrounded by a capillary network widely independently existing of the intramuscular capillary bed. The preferred innervated terminal arterioles and especially the sphincteric capillaries opening into the periganglionic capillary network and the numerous arterio-venous short-circuits in its marginal area suggest to conclude a differentiated regulation of blood supply.     

Establishment of bovine mammary epithelial cells (MAC-T): An in vitro model for bovine lactation

The hallmark of differentiated mammary epithelial cells is a copious secretion of milk-specific components regulated by lactogenic hormones. We describe an established clonal cell line produced from primary bovine mammary alveolar cells (MAC-T) by stable transfection with SV-40 large T-antigen. MAC-T cells show a population doubling time of approximately 17 h and have been cultured more than 350 passages without showing any sign of senescence. They show the characteristic “cobblestone” morphology of epithelial cells when grown on plastic substratum. Differentiation was induced by augmenting cell-cell interaction on a floating collagen gel in the presence of prolactin. The differentiated phenotype was characterized to include (1) increased abundance in β-casein mRNA, (2) increased number and size of indirect immunofluorescent casein secretory vesicles in each cell and (3) α_s- and β-casein protein secretion. The clonal nature of the cells, their immortality, and their ability to uniformly differentiate and secrete casein proteins make this cell line unique.     

Thrombin-induced events in non-platelet cells are mediated by the unique proteolytic mechanism established for the cloned platelet thrombin receptor

We recently isolated a cDNA clone encoding a functional platelet thrombin receptor that defined a unique mechanism of receptor activation. Thrombin cleaves its receptor''s extracellular amino terminal extension, unmasking a new amino terminus that functions as a tethered peptide ligand and activates the receptor. A novel peptide mimicking this new amino terminus was a full agonist for platelet secretion and aggregation, suggesting that this unusual mechanism accounts for platelet activation by thrombin. Does this mechanism also mediate thrombin''s assorted actions on non-platelet cells? We now report that the novel thrombin receptor agonist peptide reproduces thrombin-induced events (specifically, phosphoinositide hydrolysis and mitogenesis) in CCL-39 hamster lung fibroblasts, a naturally thrombin- responsive cell line. Moreover, these thrombin-induced events could be recapitulated in CV-1 cells, normally poorly responsive to thrombin, after transfection with human platelet thrombin receptor cDNA. Our data show that important thrombin-induced cellular events are mediated by the same unusual mechanism of receptor activation in both platelets and fibroblasts, very likely via the same or very similar receptors.     

Structural basis of C3b binding by glycoprotein C of herpes simplex virus.

Glycoproteins C (gC) from herpes simplex virus type 1 (HSV-1) and HSV-2, gC-1 and gC-2, bind the human complement fragment C3b, although the two glycoproteins differ in their abilities to act as C3b receptors on infected cells and in their effects on the alternative complement pathway. Previously, we identified three regions of gC-2 (I, II, and III) which are important for C3b binding. In this study, our goal was to identify C3b-binding sites on gC-1 and to continue our analysis of gC-2. We constructed a large panel of mutants by using the cloned gC-1 and gC-2 genes. Most of the mutant proteins were transported to the surface of transiently transfected L cells and reacted with one or more monoclonal antibodies to discontinuous epitopes. By using 31 linker insertion mutants spread across the coding region of gC-1, we identified four regions in the ectodomain of gC-1 which are important for C3b binding, three of which are similar in position to C3b-binding regions I, II, and III of gC-2. Region III shares some similarities with the short consensus repeat found in CR1, the human complement receptor. These were, in part, the targets for construction of 20 single amino acid changes in region III of gC-1 and gC-2. These mutants identified similarities and differences in the C3b-binding properties of gC-1 and gC-2 and suggest that the amino half of region III is more important for C3b binding. However, our results do not support the concept of a structural relationship between the short consensus repeat of CR1 and gC, since mutations of some of the conserved residues, including three of four cysteines in region III, had no effect on C3b binding. Finally, we constructed four deletion mutants of gC-1, including one which lacked residues 33 to 123, as well as residues 367 to 449. This severely truncated molecule, lacking four cysteines and five potential N-linked glycosylation sites, was transported to the cell surface and retained its ability to bind monoclonal antibodies as well as C3b. Thus, the four distinct C3b-binding regions of gC-1 and several epitopes within two different antigenic sites are localized within residues 124 to 366.     

Linkage studies in familial Alzheimer disease: Evidence for chromosome 19 linkage

A genetic component in the etiology of Alzheimer disease (AD) has been supported by indirect evidence for several years, with autosomal dominant inheritance with age-dependent penetrance being suggested to explain the familial aggregation of affecteds. St. George Hyslop et al. reported linkage of familial AD (FAD) in four early-onset families (mean age at onset [M] less than 50 years). Subsequent studies have been inconsistent in their results; Goate et al. also reported positive lod scores. However, both Pericak-Vance et al.'s study of a series of mainly late-onset FAD families (M greater than 60 years) and Schellenberg et al.'s study failed to confirm linkage to chromosome 21 (CH21). These various studies suggest the possibility of genetic heterogeneity, with some families linked to CH21 and others unlocalized. Recently, St. George Hyslop et al. extended their analysis to include additional families. The extended analyses supported their earlier finding of linkage to CH21, while showing strong evidence of heterogeneity between early-onset (M less than 65 years) and late-onset (M greater than 60 years) FAD families. Because our families did not show linkage to CH21, we undertook a genomic search for an additional locus for FAD. Because of both the confounding factor of late age at onset of FAD and the lack of clear evidence of Mendelian transmission in some of our families, we employed the affected-pedigree-member (APM) method of linkage analysis as an initial screen for possible linkage. Using this method, we identified two regions suggesting linkage: the proximal long arm of chromosome 19 (CH19) and the CH21 region of FAD linkage reported by St. George Hyslop et al. Application of standard likelihood (LOD score) analysis to these data support the possibility of an FAD gene locate on CH19, particularly in the late-onset FAD families. These data further suggest genetic heterogeneity and delineate this region of CH19 as an area needing additional investigation in FAD.     

Interim use of Jarvik-7 and Novacor artificial heart: blood rheology and transient ischemic attacks (TIA's)

The rheological properties of blood were studied in patients supported by both the Jarvik-7 total artificial heart (TAH) and Novacor left ventricular assist device (LVAD) as a bridge to cardiac transplantation. Both groups of patients had abnormalities in blood rheology which differed according to the type of device implanted as well as on the clinical state of the patient. The rheology of individual patients correlated well with their clinical status and outcome, with incidences of TIA's and/or stroke being accompanied by marked increases in relative blood viscosity, erythrocyte rigidity, fibrinogen concentration and platelet aggregation in varying combination. Observed abnormalities in blood rheology were also crucial to thrombus formation on artificial heart valves as well. Our results show that the therapeutic management of rheological parameters should prove to be a unique and clinically rewarding approach to these patients.