Effect of Vaccinium ashei reade leaves on lipid metabolism in Otsuka Long-Evans Tokushima Fatty rats

The effects of blueberry leaf (BBL) on lipid metabolism were studied in obese rats. Feeding of BBL lowered levels of serum lipids and C-reactive protein and alleviated hepatic triglyceride accumulation in the rats. The hypolipidemic effect might be attributable to a reduction of lipogenesis and enhancement of lipolysis in the liver. These results suggest the use of blueberry leaf as a dietary hypolipidemic component.     

Characterization of new milk-derived inhibitors of angiotensin converting enzyme in vitro and in vivo

Inhibition of angiotensin converting enzyme (ACE) has been observed with a variety of different peptides, and peptide fragments with inhibitory capabilities have been identified within many different proteins, including milk proteins. The purpose of this study therefore was to identify new short peptides with inhibitory properties from the primary structure of milk proteins and to characterize them in vitro and in vivo, since no milk derived ACE inhibitors have previously been evaluated for their ability to inhibit ACE in vivo. In vitro, 8 of 9 dipeptides were found to be competitive inhibitors of ACE. The IC50 was significantly lower when an angiotensin I-like substrate was used, than when a bradykinin-like substrate was used. Using three different in vivo models for ACE inhibition, a very moderate effect was observed for three of the new peptides, but only for up to 6 or 12 minutes. Nothing was observed with two reference compounds that are reported to be hypotensive ACE-inhibitors derived from milk proteins. This raises the question whether the mechanism of hypotensive action is straightforward inhibition of ACE in vivo.     

Influence of indole-3-butyric acid and propagation method on growth and development of in vitro- and ex vitro-derived lowbush blueberry plants

The effects of four indole-3-butyric acid (IBA) concentrations and two propagation methods were studied in a lowbush blueberry (Vaccinium angustifolium Ait.) clone collected from natural stands in Newfoundland and Labrador, Canada. Lowbush blueberry cultures were established in vitro from nodal explants on a modified cranberry (V. macrocarpon Ait.) tissue culture medium containing zeatin (2 μM). Blueberry plants propagated by in vitro shoot proliferation (TC) and by conventional softwood cuttings (SC) were evaluated for growth and morphology. Significant interactions for morphological characteristics were observed among the treatments. The IBA concentration had an effect on morphology of propagated plants, increasing the concentration of IBA increased stem length and leaves per stem across propagation methods. Stems per plant increased with IBA concentration up to 20 μM in SC plants, but not in TC plants. Plant vigor was affected by neither IBA concentration nor propagation method. The TC plants produced longer and more stems with more leaves per stem than the conventional cuttings. In vitro culture on zeatin-containing nutrient medium apparently induces the juvenile branching characteristics that favored enhanced vegetative growth with more stems and leaf production. It is suggested that IBA may serve as a physiologically active form of auxin in contributing to increased stem and leaf production in lowbush blueberry SC plants but not in TC plants.     

Effect of oxamethacin and sulindac on prostaglandin synthesis]

1. Indomethacin and oxamethacin reduce the formation of malonaldehyde from arachidonic acid by rat platelets in vitro. Sulindac has no influence on this formation. 2. Indomethacin, oxamethacin and sulindac inhibit the action of arachidonic acid on the rat stomach strips in vitro. 3. These three anti-inflammatory drugs suppress the hypotensive activity of arachidonic acid in the rat in vivo. 4. The comparison of the inhibitory activities of these drugs in vivo and in vitro shows that sulindac is converted to a potent anti-prostaglandin-synthetase metabolite and that a part of the action of oxamethacin depends on its conversion to indomethacin in the rat.     

Induction of Precocious Metamorphosis by 1,5-Disubstituted Imidazoles with a Non-terpene Chain

The effects of blueberry leaf (BBL) on lipid metabolism were studied in obese rats. Feeding of BBL lowered levels of serum lipids and C-reactive protein and alleviated hepatic triglyceride accumulation in the rats. The hypolipidemic effect might be attributable to a reduction of lipogenesis and enhancement of lipolysis in the liver. These results suggest the use of blueberry leaf as a dietary hypolipidemic component.     

LED light for in vitro and ex vitro efficient growth of economically important highbush blueberry (<Emphasis Type="Italic">Vaccinium corymbosum</Emphasis> L.)

Light-emitting diodes (LEDs) are useful for the growth of many plants, but not known for blueberry species. This study examined the effects of fluorescent lamps and 100 % red, 80 % red plus 20 % blue, 50 % red plus 50 % blue, and 100 % blue LEDs on the growth and development of highbush blueberry shoots under aseptic and non-aseptic conditions. Results revealed that monochromatic blue LEDs accumulated the highest contents of leaf chlorophylls. In contrast, monochromatic red LEDs inhibited chlorophyll accumulation, but produced the longest shoots and roots and provided high percentages of side shoot formation from ex vitro plants. Mixed LEDs, particularly 50 % red plus 50 % blue light, improved plant growth with respect to notably increased shoot and root biomass. Direct rooting of in vitro shoots under non-aseptic conditions was readily achieved using a commercial mixture of perlite and peat moss with high humidity controls. These findings obviously suggest the efficient use of LEDs to replace traditional fluorescent lamps in large-scale propagation of the highbush blueberry, and also pave the way for future studies on LEDs for standardizing micropropagation protocols to shrub crops and woody plants.     

Efficient in vitro regeneration systems for Vaccinium species

Efficient protocols for shoot regeneration from leaf explants suitable for micropropagation as well as for the development of transgenic plants were developed for blueberry (Vaccinium corymbosum) and lingonberry (Vaccinium vitis-idaea) cultivars. Nodal segments were used to initiate in vitro shoot cultures of lingonberry cultivar ‘Red Pearl’ and southern highbush blueberry cultivar ‘Ozarkblue’. In order to develop an optimized regeneration procedure, different types and concentrations of plant growth regulators were tested to induce adventitious shoot regeneration on excised leaves from micropropagated shoots of both cultivars. The effect on percentage regeneration and number of shoots per explant was investigated. Results indicated that zeatin was superior to TDZ and meta-topolin in promoting adventitious shoot formation. A concentration of 20 μM zeatin was most effective in promoting shoot regeneration in both cultivars, in case of ‘Red Pearl’ along with 1 μM NAA. Shoots were either allowed to root in vitro on medium containing IBA or NAA or ex vitro in a fog tunnel. IBA was superior to NAA for induction of root development in vitro in both Vaccinium cultivars. Ex vitro rooting under high humidity was tested with cuttings from mature field-grown plants, from acclimatized tissue culture derived plants and with unrooted in vitro proliferated shoots planted directly. It was found that in vitro shoots rooted better under fog than cuttings from the other plant sources and rooting was equivalent to that achieved in vitro.     

A possible alternative mechanism of kinin generation in vivo by cathepsin L

We investigated the ability of cathepsin L to induce a hypotensive effect after intravenous injection in rats and correlated this decrease in blood pressure with kinin generation. Simultaneously with blood pressure decrease, we detected plasma kininogen depletion in the treated rats. The effect observed in vivo was abolished by pre-incubation of cathepsin L with the cysteine peptidase-specific inhibitor E-64 (1 microM) or by previous administration of the bradykinin B2 receptor antagonist JE049 (4 mg/kg). A potentiation of the hypotensive effect caused by cathepsin L was observed by previous administration of the angiotensin I-converting enzyme inhibitor captopril (5 mg/kg). In vitro studies indicated that cathepsin L excised bradykinin from the synthetic fluorogenic peptide Abz-MTSVIRRPPGFSPFRAPRV-NH2, based on the Met375-Val393 sequence of rat kininogen (Abz = o-aminobenzoic acid). In conclusion, our data indicate that in vivo cathepsin L releases a kinin-related peptide, and in vitro experiments suggest that the kinin generated is bradykinin. Although it is well known that cysteine proteases are strongly inhibited by kininogen, cathepsin L could represent an alternative pathway for kinin production in pathological processes.     

Lethal and sublethal activities of imidacloprid contribute to control of adult Japanese beetle in blueberries

Field-based bioassays and residue profile analysis were used to determine the relative importance of lethal and sublethal effects of imidacloprid on adult Japanese beetle, Popillia japonica Newman, in blueberries, Vaccinium corymbosum L. Field-based bioassays assessed adult mortality and knockdown, and fruit and leaf injury from Japanese beetles exposed to 4-h and 7-d field-aged residues of imidacloprid, and the conventional insecticides azinphosmethyl and esfenvalerate. Azinphosmethyl and imidacloprid caused high levels of mortality when beetles were exposed to blueberry shoots with ripe fruit 4 h postapplication, and all compounds protected blueberry fruit and foliage from beetle feeding. Azinphosmethyl and esfenvalerate caused significant Japanese beetle mortality when adults were exposed to blueberry shoots 7 d postapplication, whereas imidacloprid residues caused effects that protected leaves, although not of ripe fruit. When beetles were exposed to shoots with immature green fruit, relatively more leaf feeding and mortality were observed, suggesting that earlier treatment timings may be most effective for systemic neonicotinoids. Japanese beetle mortality was highly correlated with imidacloprid fruit and leaf surface residues, whereas sublethal feeding deterrent effects were observed after the surface residues diminished. The value of the plant-insect-chemistry model for describing the spatial and temporal dimensions of insecticide modes of activity is discussed in terms of optimizing crop protection.     

L-arginine availability determines the duration of acetylcholine-induced systemic vasodilation in vivo

In vitro studies have shown that acetylcholine-induced vasorelaxation is mediated by endothelium-derived relaxing factor/nitric oxide (EDRF/NO). EDRF/NO is synthesized from L-arginine by an enzymatic pathway that is inhibited by L-NG-methylarginine. To assess whether EDRF/NO also mediates the vasodilating action of acetylcholine in vivo, we have investigated the effect of L-arginine and L-NG-methylarginine on the hypotensive response to acetylcholine in the anesthetized guinea pig. L-arginine prolonged the duration of the depressor response to acetylcholine and L-NG-methylarginine decreased it. However, neither L-arginine nor L-NG-methylarginine modified the magnitude of acetylcholine's hypotensive effect unless the blood pressure was previously elevated by infusion with norepinephrine. Thus, de novo synthesis of nitric oxide from L-arginine contributes importantly, but not exclusively, to acetylcholine's hypotensive effect in the guinea pig. Furthermore, the concentration of circulating L-arginine may influence the duration and magnitude of acetylcholine-induced depressor responses under normotensive and hypertensive conditions.     

Sucrose level influences micropropagation and gene delivery into leaves from in vitro propagated highbush blueberry shoots

In an effort to increase in vitro blueberry (Vaccinium corymbosum L.) shoot production without negatively impacting subsequent genetic engineering experiments, studies were conducted to examine the effects of sucrose concentration in the propagation medium on shoot proliferation and on the transfer of an intron-containing -glucuronidase (GUS) gene into leaf explants from the propagated shoots. Numbers of axillary shoots >0.5 cm in length did not significantly increase for `Bluecrop' when sucrose levels were increased from 15 mM to either 29, 44 or 58 mM. The number of axillary shoots increased significantly for Duke ' and `Georgiagem' when sucrose concentrations were increased from 15 to 44 mM, and from 15 to 58 mM, respectively. Four-days of cocultivation with Agrobacterium tumefaciens strain EHA105 yielded highest GUS-expressing leaf zones on leaf explants from shoots cultured on either 15 or 29 mM sucrose. The number of GUS-expressing leaf zones was significantly lower on leaf explants derived from shoots grown on 58 mM sucrose than from those grown on 15 mM sucrose for all three cultivars, and was significantly lower on 44 mM compared to 15 mM for cultivars Duke and Georgiagem. These studies indicate shoot pretreatment conditions for optimizing subsequent blueberry genetic engineering experiments. Thus, a blueberry shoot proliferation medium containing 15–29 mM sucrose is recommended for explants later used for genetic transformation.     

Detection of DNA methylation pattern in thidiazuron-induced blueberry callus using methylation-sensitive amplification polymorphism

During the normal developmental process, programmed gene expression is an essential phenomenon in all organisms. In eukaryotes, DNA methylation plays an important role in the regulation of gene expression. The extent of cytosine methylation polymorphism was evaluated in leaf tissues collected from the greenhouse grown plants and in in vitro-derived callus of three lowbush and one hybrid blueberry genotypes, using methylation-sensitive amplification polymorphism (MSAP) technique. Callus formation started from the leaf segments after 4 weeks of culture on a thidiazuron (TDZ) containing medium. Maximum callus formation (98 %) was observed in the hybrid blueberry at 1.0 mg dm^-3 TDZ. Although noticeable changes in cytosine methylation pattern were detected within the MSAP profiles of both leaf and callus tissues, methylation events were more polymorphic in calli than in leaf tissues. The number of methylated CCGG sites varied significantly within the genotypes ranging from 75 to 100 in leaf tissues and from 215 to 258 in callus tissues. Differences in the methylation pattern were observed not only in a tissue-specific manner but also within the genotype in a treatment specific manner. These results demonstrated the unique effect of TDZ and the tissue culture process on DNA methylation during callus development.     

Role of platelets in arterial hypotension induced by arachidonic acid and in carrageenan induced edema in the rat]

When rat platelets are incubated in vitro in the presence of aspirin, the formation of malonaldehyde from arachidonic acid is inhibited. The production of malonaldehyde reflects the synthesis of prostaglandins and associated compounds. The same inhibition is found when the platelets originate from rats pretreated with aspirin. Small doses of aspirin are active in vitro and 10-20 mg/kg in vivo. This dosage of aspirin does not affect the hypotensive activity of arachidonic acid nor the oedematous properties of carrageenan in the rat. These two effects are thus independent from the prostaglandins formed in the platelets.     

Polyphenolics enhance red blood cell resistance to oxidative stress: in vitro and in vivo

In this study we investigated the potential antioxidant properties of blueberry polyphenolics in vitro and vivo, using red blood cell (RBC) resistance to reactive oxygen species (ROS) as the model. In vitro incubation with anthocyanins or hydroxycinnamic acids (HCA) (0.5 and 0.05 mg/ml) was found to enhance significantly RBC resistance to H2O2 (100 microM) induced ROS production. This protection was also observed in vivo following oral supplementation to rats at 100 mg/ml. However, only anthocyanins were found to afford protection at a significant level, this at 6 and 24 h post supplementation. This protection was not consistent with the measured plasma levels of anthocyanins. Indeed, plasma polyphenolic concentrations were highest after 1 h, declining considerably after 6 h and not detected after 24 h. The difference in absorption between anthocyanins and HCA is likely to have contributed to the observed difference in their abilities to afford protection to RBC. This protection represents a positive role following dietary consumption of polyphenolics from blueberries, against ROS formation within RBC in vivo.     

Effect of moxonidine on putative sympathetic neurons in the rostral ventrolateral medulla of the rat

We used an intracellular recording technique in vitro to investigate the effects of moxonidine on neurons in the rostral ventrolateral medulla (RVLM) with electrophysiological properties similar to premotor sympathetic neurons in vivo. These neurons were classified as firing regularly and irregularly, according to previous reports. Moxonidine is a sympathoinhibitory and antihypertensive agent that is thought to be a ligand of alpha(2)-adrenergic receptors and imidazoline type-1 receptors in the RVLM. Moxonidine (2-10 microM) was applied to the perfusate on 4 irregularly firing neurons, and 2 regularly firing neurons. Moxonidine (2 microM) produced only minor depolarization in 2 of these neurons. However, on 4 tested neurons, moxonidine (10 microM) elicited a profound inhibitory effect with hyperpolarization (near -20 mV); these neurons practically ceased firing. These changes were partially reversible. The results would indicate that neurons in the RVLM, recorded in vitro and with similar electrophysiological characteristics to a group of neurons previously identified in vivo in the same bulbar region as barosensitive premotor sympathetic neurons, can be modulated by imidazoline-derivative adrenergic agonists. These results could help to understand the hypotensive effects of moxonidine.     

Cyproheptadine as an inhibitor of the effects of bradykinin]

A study was made of the influence of ciproheptadin, serotonin and histamine inhibitor, on the effects of exo- and endogenous bradykinin. Ciproheptadin inhibited the effect of bradykinin on a section of a guinea pig intestine in vitro, eliminated the hypotensive effect of bradykinin in rats in vivo, and also blocked the constriction of the vessels of the ear in rabbit and a fall of the blood kininogen level caused by the administration of pyrogenal.     

Development of 3,4-dihydro-2H-benzo[1,4]oxazine derivatives as dual thromboxane A2 receptor antagonists and prostacyclin receptor agonists

We discovered a novel series of 3,4-dihydro-2H-benzo[1,4]oxazin-8-yloxyacetic acid derivatives as potent dual-acting agents to block the TXA2 receptor and to activate the PGI2 receptor. We report the synthesis, structure-activity relationship, and in vitro, ex vivo, and in vivo pharmacology of this series of compounds. 4-[2-(1,1-Diphenylethylsulfanyl)ethyl]-3,4-dihydro-2H-benzo[1,4]oxazin-8-yloxyacetic acid N-methyl-D-glucamine salt (7) is a promising candidate for a novel treatment in the anti-thrombotic and the cardiovascular fields avoiding hypotensive side effects.     

Infection of wild and cultivated Vaccinium spp. with blueberry leaf mottle nepovirus

A field survey was performed during the summers of 1991 and 1992 to determine whether blueberry leaf mottle virus (BLMV) infects wild blueberry bushes surrounding commercial blueberry fields and might act as a reservoir of the virus. Leaf samples from wild blueberries growing in wooded areas surrounding commercial plantings in Western central Lower Michigan were taken from bushes radiating out from the border of the commercial field into the wooded areas at 5, 50 and 100 m. BLMV was present in native bushes of Vaccinium corymbosum, V. myrtilloides and V. angustifolium, and two hybrids of V. corymbosum V. angustifolium. BLMV-infected pollen grains were detected in pollen traps located up to 2 miles from a source field.     

Atrial natriuretic peptide decreases cardiac output independent of coronary vasoconstriction

Studies were performed in isolated, Langendorff-perfused rat hearts and anesthetized dogs to determine the effects of synthetic atrial natriuretic peptide (ANP 8-33) on the coronary circulation. In vitro studies in the rat examined coronary flow dynamics to ANP 8-33 over a defined range from physiologic to pharmacologic concentrations. No changes in coronary flow or chronotropic and inotropic function of the isolated Langendorff-perfused heart were observed in response to increasing concentrations of ANP 8-33 (10(2) to 10(6) pg/ml). In the dog, a low, nonhypotensive dose of ANP 8-33 (0.05 microgram/kg/min) decreased cardiac output with no change in coronary blood flow or coronary vascular resistance. At a high, hypotensive dose (0.3 microgram/kg/min) ANP 8-33 decreased cardiac output in association with transient coronary vasodilation. Continued infusion resulted in a decrease in coronary blood flow and arterial pressure with no change in coronary vascular resistance. Thus, in vitro physiologic and pharmacologic concentrations of ANP, or in vivo low concentrations of ANP, do not result in an alteration in coronary flow. In vivo ANP 8-33, at both nonhypotensive and hypotensive concentrations, decreased cardiac output in the absence of coronary vasoconstriction.