Biochemical evidence for a calcium-dependent protein kinase from Pharbitis nil and its involvement in photoperiodic flower induction

A soluble Ca(2+)-dependent protein kinase (CDPK) was isolated from seedlings of the short-day plant Pharbitis nil and purified to homogeneity. Activity of Pharbitis nil CDPK (PnCDPK) was strictly dependent on the presence of Ca(2+) (K(0,5)=4,9 microM). The enzyme was autophosphorylated on serine and threonine residues and phosphorylated a wide diversity of substrates only on serine residues. Histone III-S and syntide-2 were the best phosphate acceptors (K(m) for histone III-S=0,178 mg ml(-1)). Polyclonal antibodies directed to a regulatory region of the soybean CDPK recognized 54 and 62 kDa polypeptides from Pharbitis nil. However, only 54 kDa protein was able to catalyse autophosphorylation and phosphorylation of substrates in a Ca(2+)-dependent manner. CDPK autophosphorylation was high in 5-day-old Pharbitis nil seedlings grown under non-inductive continuous white light and was reduced to one-half of its original when plants were grown in the long inductive night. Also, the pattern of proteins phosphorylation has changed. After 16-h-long inductive night phosphorylation of endogenous target (specific band of 82 kDa) increased in the presence of calcium ions. It may suggest that Ca(2+)-dependent protein kinase is involved in this process and it is dependent on light/dark conditions.     

Pharmacological activity of the essential oil of Satureja viminea (Lamiaceae)

The aqueous extract and the essential oil of Satureja viminea (Lamiaceae) were tested. General physiologic effects were assessed through the Hippocratic screening test. Non fasted female Sprague Dawley rats were utilized and 250, 500, 750 and 1000 mg/kg doses were used. Two animals were used for each dosage level and for the vehicle alone. Exploratory behavior and curiosity were measured using a hole board apparatus and placing non-trained mice on the board and recording the number of holes explored in a 5 minute period. The Boissier chimney test was used to evaluate motor coordination. Muscle strength was assessed through a grasping test where mice were hung by their fore-limbs 40 cm above the base on a horizontal metal stainless bar. In all these tests, 3 groups of 6 albino mice, were treated with 1000 mg/kg of each the essential oil of S. viminea, the vehicle and diazepan (1 mg/kg) as a positive control. Analgesic activity was explored in Sprague-Dawley rats. The tail flick method described by D'Amour and Smith (1941) modified by CYTED was implemented on three groups (6 rats each) of animals treated with, each the essential oil of S. viminea (1000 mg/kg), the vehicle and indomethacine. The test was carried out just before and 30, 60 and 120 min after oral treatment. Peristaltic activity was measured in albino mice, three groups of 6 animals each, treated orally with each the essential oil of S. viminea (1000 mg/kg), the aqueous extract (1000 mg/kg), and the vehicle. The marker used was activated carbon. Animals were sacrificed 30 min after the marker was given and the percent of total small intestine traversed by it was calculated. Also a lethal dose 50 (LD 50) was determined with the Spearman-Karber method. A dose-related spontaneous motor activity reduction was observed. Exploratory behavior and curiosity were diminished. The grasping strength of mice was reduced. A very clear and significant analgesic effect was observed with the oral administration of the essential oil of S. viminea (1000 mg/kg). This effect is compared to that of indomethacine. Intestinal transit and gastric emptying were inhibited by the essential oil. The LD50 of the essential oil of S. viminea is 556.8 mg/kg.     

The guanylate binding protein-1 GTPase controls the invasive and angiogenic capability of endothelial cells through inhibition of MMP-1 expression

Expression of the large GTPase guanylate binding protein-1 (GBP-1) is induced by inflammatory cytokines (ICs) in endothelial cells (ECs), and the helical domain of the molecule mediates the repression of EC proliferation by ICs. Here we show that the expression of GBP-1 and of the matrix metalloproteinase-1 (MMP-1) are inversely related in vitro and in vivo, and that GBP-1 selectively inhibits the expression of MMP-1 in ECs, but not the expression of other proteases. The GTPase activity of GBP-1 was necessary for this effect, which inhibited invasiveness and tube-forming capability of ECs in three-dimensional collagen-I matrices. A GTPase-deficient mutant (D184N-GBP-1) operated as a transdominant inhibitor of wild-type GBP-1 and rescued MMP-1 expression in the presence of ICs. Expression of D184N-GBP-1, as well as paracrine supplementation of MMP-1, restored the tube-forming capability of ECs in the presence of wild-type GBP-1. The latter finding indicated that the inhibition of capillary formation is specifically due to the repression of MMP-1 expression by GBP-1, and is not affected by the anti-proliferative activity of the helical domain of GBP-1. These findings substantiate the role of GBP-1 as a major regulator of the anti-angiogenic response of ECs to ICs.     

The structure of ActVA-Orf6, a novel type of monooxygenase involved in actinorhodin biosynthesis

ActVA-Orf6 monooxygenase from Streptomyces coelicolor that catalyses the oxidation of an aromatic intermediate of the actinorhodin biosynthetic pathway is a member of a class of small monooxygenases that carry out oxygenation without the assistance of any of the prosthetic groups, metal ions or cofactors normally associated with activation of molecular oxygen. The overall structure is a ferredoxin-like fold with a novel dimeric assembly, indicating that the widely represented ferredoxin fold may sustain yet another functionality. The resolution (1.3 A) of the enzyme structure and its complex with substrate and product analogues allows us to visualize the mechanism of binding and activation of the substrate for attack by molecular oxygen, and utilization of two gates for the reaction components including a proton gate and an O(2)/H(2)O gate with a putative protein channel. This is the first crystal structure of an enzyme involved in the tailoring of a type II aromatic polyketide and illustrates some of the enzyme-substrate recognition features that may apply to a range of other enzymes involved in modifying a polyketide core structure.     

Relationship between the action of reactive oxygen and nitrogen species on bilayer membranes and antioxidants

Membrane lipid peroxidation (LPO) induced by hydroxyl (*OH) and ascorbyl (*Asc) radicals and by peroxynitrite (ONOO-) was investigated in asolectin (ASO), egg phosphatidylcholine (PC) and PC/phosphatidic acid mixtures (PC:PA) liposomes and rat liver microsomes (MC). Enthalpy variation (DeltaH) of PC:PA at different molar ratios were obtained by differential scanning calorimetry. It was also evaluated the LPO inhibition by quercetin, melatonin and Vitamin B6. The oxidant effect power follows the order *OH approximately *Asc > ONOO- on PC and MC; whilst on ASO liposomes, it follows *Asc > *OH approximately ONOO-. Increasing amounts of PA in PC liposomes resulted in lower levels of LPO. The DeltaH values indicate a more ordered membrane arrangement as a function of PA amount. The results were discussed in order to provide a complete view involving the influence of membranes, oxidants and antioxidants intrinsic behavior on the LPO dynamics.     

Effect of lipopolysaccharides on human dendritic cell cultures and its inhibition by polymyxin B

Dendritic cells have been described as effective antigen presenting cells. Human dentritic cells are highly susceptible to lipopolysaccharide (LPS) tolerance, consisting of a differential deactivation state in which some cellular functions are impaired. LPS tolerance can be experimentally induced in vitro, in which the presence of LPS strongly affects the behavior of cultured dendritic cells. Recombinant proteins obtained from bacterial systems or protein extracts of ectoparasites containing LPS can be used as stimuli to enhance maturation processes in these cells. The present study evaluated the effect of LPS in human dendritic cell cultures, and the activity of polymyxin B as an inhibitor of the LPS effect. Dendritic cells were obtained from peripheral blood monocytes in the presence of IL-4 and GM-CSF, followed by exposure with LPS and PGE2/TNFalpha. Surface markers and cytokine levels were evaluated by flow cytometry. The dendritic cells pre-exposed to single doses of endotoxin demonstrated a reduced capacity to mature, reduced CD83 expression, inhibited secretion of IL-12, TNFalpha, IL-10 and diminished secretion of IL-6. Furthermore, polymyxin B at 10 mg/ml inhibits LPS activity at 1 mg/ml. The maximum polymyxin B concentration with no effect on cellular morphology was 50 mg/ml. Consequently, polymyxin B was determined to be an effective LPS inhibitor in dendritic cell cultures.     

Correlation between the physicochemical properties of organic solvents and their biocompatibility toward epoxide hydrolase activity in whole-cells of a yeast, Rhodotorula sp.

Epoxides are often highly hydrophobic substrates and the presence of an organic co-solvent within an aqueous bioreactor is in such cases indicated. The effect of 40 water-miscible and -immiscible organic solvents on epoxide hydrolase activity in whole-cells of the yeast Rhodotorula sp. UOFS Y-0448 was investigated. No formal correlation between solvent biocompatibility and physicochemical properties was deductible, although the introduction of hydroxyl groups increased biocompatibility. 1-Pentanol, 2-methylcyclohexanol and 1-octanol were the most biocompatible resulting in relatively low activity losses when used at up to 20% (v/v).     

Effect of thyroid hormones on the gene expression of calcium transport systems in rat muscles

It has been previously shown that modification of thyroid hormone levels have a profound impact on cardiac function, predominantly through a direct regulation of the sarcoplasmic reticulum protein levels. Nevertheless, little is known about the regulation of calcium transport systems in skeletal muscle due to the altered concentration of thyroid hormones. Thus, the goal of our study was to find out whether altered thyroid status could change the gene expression of the Na(+)/Ca(2+) exchanger (NCX), the inositol 1,4,5-trisphosphate (IP(3)) receptors and ryanodine receptors (RyRs) in slow and fast skeletal muscles of rats. A hyperthyroid state was maintained in rats by triiodothyronine (T(3)) administration, while methimazole was employed for inducing hypothyroidism. After a period of 2-10 months of T(3) treatment we observed a significant increase in mRNA levels of the NCX, RyRs and IP(3) receptors. This increase was more pronounced in the slow soleus than in the fast extensor digitorum longus (EDL) muscle. It is tempting to speculate that thyroid hormones also alter calcium concentration and thus influence the process of excitation-contraction coupling in the skeletal muscle.