Purification and partial characterization of serum monocytotropic factor, a platelet-derived cyclooxygenase-inducing polypeptide

It has previously been shown that a heat- and acid-stable component of human and animal sera was capable of stimulating prostanoid biosynthesis in human blood monocytes, very probably by a mechanism involving cyclooxygenase induction. Many physico-chemical characteristics of this factor are similar to those of identified platelet factors. Here we show that human platelets are a rich source of this factor (serum monocytotropic factor) and that results from experiments using arachidonic acid or thrombin as releasers are consistent with its presence in platelet membranes. Serum monocytotropic factor has been purified 1500-fold by three chromatographic steps. Purification was more difficult when starting from platelet releasates or lysates. The purified serum monocytotropic factor had an apparent molecular mass of 70,000 as judged by Sephadex G-75 chromatography and by polyacrylamide gel electrophoresis; however, when subjected to HPLC on a gel permeation column in the presence of 6 M urea, one major peak corresponding to a relative molecular mass (Mr) of 30,000-35,000 was observed, which suggests a homodimeric structure. It is therefore very likely that human platelets store, in addition to the two well-identified polypeptide growth factors, platelet-derived growth factor and transforming growth factor-beta, a third polypeptide capable of regulating prostanoid production in monocytes.     

Regulation of thromboxane A2 biosynthesis in platelet-free human monocytes and the possible role of polypeptide growth factor(s) in the induction of cyclooxygenase system

It has previously been shown that platelet-free human monocytes, when properly incubated in the presence of animal and human sera, became capable of producing large amounts of thromboxane A2 and prostaglandin E2. The characteristics of these processes are reported here. Prostaglandin biosynthesis was time and cell concentration dependent; 24 h of incubation at 37 degrees C and 0.5 X 10(6) cells per ml medium were found to give the most reproducible results. Human monocytes produced thromboxane A2 and prostaglandin E2 in a typical ratio which ranged from 2.0 to 5.0 (28 experiments). Animal and human sera were similarly effective, while serum obtained from platelet-free blood was much less active. The activity of all sera tested was stable to heating (100 degrees C for 2-10 min) and extreme pH values (pH 2 and 11). It was unstable when the serum was heated at pH 11 and after 2-mercaptoethanol treatment. These observations prompted us to check the effect of polypeptide growth factors having properties similar to those reported above, such as platelet-derived growth factor, fibroblast growth factor, epidermal growth factor as well as insulin and transferrin. None of these, alone or in various combinations, was capable of eliciting a stimulation comparable with that of serum. Stimulation due to sera was, as expected, dose dependently inhibited by acetylsalicylic acid and more efficiently by indomethacin; unexpectedly it was also inhibited by protein synthesis inhibitors such as actinomycin D and cycloheximide in conditions under which no toxic effect of the drugs was evident. On the basis of these results we conclude that: (a) polypeptide growth factor(s) with a molecular weight at least 30 000 (as judged by Amicon ultrafiltration) is involved in the regulation of prostaglandin biosynthesis); (b) such a factor(s) acts by inducing rather than by activating the cyclooxygenase system.     

Decrease in the Function of the γ-Aminobutyric Acid-Coupled Chloride Channel Produced by the Repeated Administration of Pentylenetetrazol to Rats

The acute administration of pentylenetetrazol (PTZ; 25-75 mg/kg i.p.) failed to modify the specific binding of t-[35S]butylbicyclophosphorothionate ([35S]TBPS) to membrane preparations from the cerebral cortex of the rat. In contrast, the repeated administration of PTZ (30 mg/kg i.p., three times a week for 12 weeks) reduced by 26% the density of [35S]TBPS binding sites without modifying the dissociation constant. This effect was observed 3 days after the last PTZ administration. A parallel reduction of gamma-aminobutyric acid (GABA)-stimulated 36Cl- uptake was measured in the cerebral cortex of PTZ-treated rats 3 days after the last injection. The repeated administration of PTZ produced sensitization to the drug, or chemical kindling. In fact, no convulsions were observed in the first week of treatment, but all the animals became sensitized to PTZ by the 12th week. The results are consistent with the hypothesis that chronic treatment with PTZ at a subconvulsant dose causes a decrease in GABA-coupled chloride channel activity that may be related to the chemical kindling produced by this compound.     

Kinetics of tert-[35S]Butylbicyclophosphorothionate Binding in the Cerebral Cortex of Newborn and Adult Rats: Effects of GABA and Receptor Desensitization

Abstract: The effects of GABA on the kinetics of tert -[³⁵S]butylbicyclophosphorothionate ([³⁵S]TBPS) binding to the convulsant site of GABA_A receptors were studied in membrane suspensions from the cerebral cortex of newborn (1-day-old) and adult (90-day-old) rats. TBPS dissociation was biphasic in neonates and adults, indicating that more than one interconvertible state of [³⁵S]TBPS binding sites may be present in the cerebral cortex. In the absence of GABA, the fast ( t _1/2, 11 min) and slow ( t _1/2, 77 min) components of TBPS dissociation in newborn rats were approximately fourfold slower than in adults. The acceleration of the dissociation rates caused by 2 µ M GABA, however, was more robust in neonates than in adults (six- to ninefold vs. twofold increase, respectively). Moreover, the dissociation rates of TBPS in membranes preincubated with 2 µ M GABA (dissociation started by adding 40 µ M picrotoxin) were two- to fourfold slower than in membranes preincubated without GABA (dissociation started by adding 40 µ M picrotoxin plus 2 µ M GABA). Taken together, these results suggest that (1) the closed state of GABA_A receptors is associated with a more effective steric barrier for the binding of TBPS in neonates compared with adults, (2) GABA produces a larger acceleration of the binding kinetics of TBPS in neonates than in adults, and (3) long incubations with GABA may cause receptor desensitization, which in turn slows down the dissociation rates of TBPS.     

A 10-miRNA risk score-based prediction model for pathological complete response to neoadjuvant chemotherapy in hormone receptor-positive breast cancer

Patients with hormone receptor(HR)-positive tumors breast cancer usually experience a relatively low pathological complete response(p CR) to neoadjuvant chemotherapy(NAC). Here, we derived a 10-micro RNA risk score(10-mi RNA RS)-based model with better performance in the prediction of p CR and validated its relation with the disease-free survival(DFS) in 755 HRpositive breast cancer patients(273, 265, and 217 in the training, internal, and external validation sets, respectively). This model,pres...     

Olive flowering trends in a large Mediterranean area (Italy and Spain)

The aim of this study was to investigate the main climatic and biological trends related to olive flowering in central-southern Italy compared to those in Andalusia, Spain. Results since 1982 were compared for the two long-series monitoring areas of Cordoba and Perugia, and since 1992–1999 for the short-series areas. The relationship between climatic trends and the biological response of the olive, a widespread culture in the Mediterranean basin, were investigated. An aerobiological method involving capturing pollen released into the atmosphere was utilised as a bioindicator of flowering phenology. The study results confirm the strong relationship between flowering periods and spring temperature trends for the olive. Temperature during March, April and May was the parameter most related to flowering date in the study areas, particularly in Italy. In some cases we found a significant correlation between flowering and past autumn temperatures, probably due to their effect on floral bud dormancy induction, but this phenomenon appeared to be of minor importance in the studied areas. The phenological trend results show the continuous advance of flowering dates to the late 1990s, followed by a relatively stationary time series related to a short-term temperature fluctuation in the Mediterranean area. This latter period probably represents a mesoscale event forced by a macroscale event—the North Atlantic Oscillation. The results reveal that the trend towards increased temperatures, and the consequent flowering advance of some species, indicated some years ago is nowadays not as clear as was expected and should be confirmed over the next few years in the Mediterranean areas under investigation.