16a-METHYL CORTICOSTEROIDS—A New Series of Anti-Inflammatory Compounds; Clinical Appraisal of Their Antirheumatic Potencies

Four new derivatives of hydrocortisone, each containing in common a methyl grouping at the 16a-carbon position of the steroid molecule, have been synthesized and are being studied in human subjects. The compounds are 16a-methyl 9a-fluoroprednisolone (MK-125: hexadecadrol), 16a-methyl 9a-fluorohydrocortisone (MK-126), 16a-methylprednisolone (MK-110), and 16a-methylhydrocortisone (MK-117). Biologic tests in animals have indicated that these analogues exhibit, in varying degrees, striking alterations of several physiologic properties, including enhanced anti-inflammatory activity unassociated with corresponding disturbance of electrolyte metabolism.In the present study preliminary observations of the effects of the four new compounds were made in patients with rheumatoid arthritis. Clinical estimates of the antirheumatic potencies of the compounds, as compared with prednisolone, were accomplished by determining the milligram dosages required to maintain similar degrees of improvement of active rheumatoid manifestations. The approximate antirheumatic potencies of the compounds, on an average, were gauged as follows: for 16a-methyl 9a-fluoroprednisolone, about seven times greater than prednisolone; for 16a-methyl 9a-fluorohydrocortisone, about three times greater; for 16a-methylprednisolone, approximately one-third greater; and for 16a-methylhydrocortisone, about 70 per cent that of prednisolone. In the dosage used, none of the compounds promoted discernible salt and water retention.These observations would indicate that 16a-methyl 9a-fluoroprednisolone (hexadecadrol) possesses greater anti-inflammatory potency per milligram than any steroid yet produced. The therapeutic efficiency of the compound on longterm administration is being studied.     

Ground wētā in vines of the Awatere Valley,Marlborough: biology,density and distribution

The endemic New Zealand ground wētā (Hemiandrus sp. ‘promontorius’) has a Naturally Uncommon conservation status. This is because of the paucity of information on its density and distribution. Here, the biology, density and distribution of a population of this wētā found in and around vineyards in the Awatere Valley, Marlborough was studied. Wētā density was assessed in vineyards, paddocks and shrublands in this valley. Soil moisture, penetration resistance, pH and organic matter were recorded at locations with and without wētā. Wētā density in vineyards was significantly higher than in either paddocks or shrub habitats. In vineyards, the density of this insect was significantly higher under-vines than in the inter-rows. Higher numbers of this wētā were found in moist soils that required lower force to burrow. Females laid an average of 55 eggs between March and April, which hatched in September. These findings highlight the intersection between agriculture and conservation.     

Timing of the first cleavage post‐insemination affects cryosurvival of in vitro–produced bovine blastocysts

The time of the first cleavage of bovine zygotes during in vitro culture can affect the rate of development and cell number of the blastocysts. The aim of this work was to study the effect of the timing of first cleavage on the cryosurvival of the resulting blastocysts. Following standard IVM and IVF, zygotes were cultured in modified synthetic oviduct fluid (SOF), with 10% fetal calf serum (FCS) added 48 hr post insemination, in a humidified atmosphere of 5% CO_2, 5% O₂ and 90% N₂. Embryos which cleaved by 24, 27 30, 33, or 36 hr after insemination (IVF) were harvested and further cultured to the blastocyst stage (day 7 or day 8 post IVF). All developing blastocysts on days 7 and 8 were classified into three groups and were cryopreserved by vitrification. Group A consisted of blastocysts (<150 μm, small blastocysts); group B consisted of expanded or hatching blastocysts (>150 μm, large blastocysts); and group C consisted of morphologically poor quality blastocysts. The vitrification solution consisted of 6.5 M glycerol and 6% bovine serum albumin in PBS (VS3a). Thawed embryos were cultured further and survival was defined as the re‐expansion and maintenance of the blastocoel over 24, 48, and 72 hr, respectively. Overall survival and hatching at 72 hr post‐thawing was higher in blastocysts formed by day 7 than those formed by day 8 (60% vs. 40% survival; 63% vs. 45% hatching). Large blastocysts from day‐7 and day‐8 groups survived significantly better than small or poor quality blastocysts (76% vs. 63% and 31%; 72% vs. 30% and 26%, respectively; P < 0.05). Day‐7 blastocysts from the 27‐ and 30‐hr cleavage groups survived significantly better than those from the 36‐hr group (63% and 66% vs. 25%, P < 0.05). Day‐8 blastocysts from later cleaved (30 hr) zygotes had a higher survival than the 27‐hr cleavage groups (52% vs. 26%, P < 0.05). These results indicate that the day of blastocyst appearance, developmental stage, and timing of the first cleavage post‐insemination can influence the cryosurvival of bovine blastocysts following vitrification. Mol. Reprod. Dev. 53:318–324, 1999. © 1999 Wiley‐Liss, Inc.     

Implication of HMGR in homeostasis of sequestered and de novo produced precursors of the iridoid biosynthesis in leaf beetle larvae

Insects employ iridoids to deter predatory attacks. Larvae of some Chrysomelina species are capable to produce those cyclopentanoid monoterpenes de novo. The iridoid biosynthesis proceeds via the mevalonate pathway to geranyl diphospate (GDP) subsequently converted into 8-hydroxygeraniol-8-O-beta-D-glucoside followed by the transformation into the defensive compounds. We tested whether the glucoside, its aglycon or geraniol has an impact on the activity of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), the key regulatory enzyme of the mevalonate pathway and also the iridoid biosynthesis. To address the inhibition site of the enzyme, initially a complete cDNA encoding full length HMGR was cloned from Phaedon cochleariae. Its catalytic portion was then heterologously expressed in Escherichia coli. Purification and characterization of the recombinant protein revealed attenuated activity in enzyme assays by 8-hydroxygeraniol whereas no effect has been observed by addition of the glucoside or geraniol. Thus, the catalytic domain is the target for the inhibitor. Homology modeling of the catalytic domain and docking experiments demonstrated binding of 8-hydroxygeraniol to the active site and indicated a competitive inhibition mechanism. Iridoid producing larvae are potentially able to sequester glucosidically bound 8-hydroxygeraniol whose cleavage of the sugar moiety results in 8-hydroxygeraniol. Therefore, HMGR may represent a regulator in maintenance of homeostasis between de novo produced and sequestered intermediates of iridoid metabolism. Furthermore, we demonstrated that HMGR activity is not only diminished in iridoid producers but most likely prevalent within the Chrysomelina subtribe and also within the insecta.     

Effects of feeding Spodoptera littoralis on lima bean leaves: IV. Diurnal and nocturnal damage differentially initiate plant volatile emission

Continuous mechanical damage initiates the rhythmic emission of volatiles in lima bean (Phaseolus lunatus) leaves; the emission resembles that induced by herbivore damage. The effect of diurnal versus nocturnal damage on the initiation of plant defense responses was investigated using MecWorm, a robotic device designed to reproduce tissue damage caused by herbivore attack. Lima bean leaves that were damaged by MecWorm during the photophase emitted maximal levels of beta-ocimene and (Z)-3-hexenyl acetate in the late photophase. Leaves damaged during the dark phase responded with the nocturnal emission of (Z)-3-hexenyl acetate, but with only low amounts of beta-ocimene; this emission was followed by an emission burst directly after the onset of light. In the presence of (13)CO(2), this light-dependent synthesis of beta-ocimene resulted in incorporation of 75% to 85% of (13)C, demonstrating that biosynthesis of beta-ocimene is almost exclusively fueled by the photosynthetic fixation of CO(2) along the plastidial 2-C-methyl-D-erythritol 4-P pathway. Jasmonic acid (JA) accumulated locally in direct response to the damage and led to immediate up-regulation of the P. lunatus beta-ocimene synthase gene (PlOS) independent of the phase, that is, light or dark. Nocturnal damage caused significantly higher concentrations of JA (approximately 2-3 times) along with enhanced expression levels of PlOS. Transgenic Arabidopsis thaliana transformed with PlOS promoter :: beta-glucuronidase fusion constructs confirmed expression of the enzyme at the wounded sites. In summary, damage-dependent JA levels directly control the expression level of PlOS, regardless of light or dark conditions, and photosynthesis is the major source for the early precursors of the 2-C-methyl-D-erythritol 4-P pathway.     

Extracellular ATP activates MAP kinase cascades through a P2Y purinergic receptor in the human intestinal Caco-2 cell line

Background

ATP exerts diverse effects on various cell types via specific purinergic P2Y receptors. Intracellular signaling cascades are the main routes of communication between P2Y receptors and regulatory targets in the cell.

Methods and results

We examined the role of ATP in the modulation of ERK1/2, JNK1/2, and p38 MAP kinases (MAPKs) in human colon cancer Caco-2 cells. Immunoblot analysis showed that ATP induces the phosphorylation of MAPKs in a time- and dose-dependent manner, peaking at 5 min at 10 µM ATP. Moreover, ATPγS, UTP, and UDP but not ADP or ADPβS increased phosphorylation of MAPKs, indicating the involvement of, at least, P2Y₂/P2Y₄ and P2Y₆ receptor subtypes. RT–PCR studies and PCR product sequencing supported the expression of P2Y₂ and P2Y₄ receptors in this cell line. Spectrofluorimetric measurements showed that cell stimulation with ATP induced transient elevations in intracellular calcium concentration. In addition, ATP-induced phosphorylation of MAPKs in Caco-2 cells was dependent on Src family tyrosine kinases, calcium influx, and intracellular Ca²⁺ release and was partially dependent on the cAMP/PKA and PKC pathways and the EGFR.

General significance

These findings provide new molecular basis for further understanding the mechanisms involved in ATP functions, as a signal transducer and activator of MAP kinase cascades, in colon adenocarcinoma Caco-2 cells.