Stimulants of prostaglandin biosynthesis in human fetal membranes, uterine decidua vera and placenta

Modulation of prostaglandin (PG) biosynthesis by cytosolic fractions derived from homogenates of human amnion, chorion laeve, decidua vera and placenta was examined. PGF_2α and 6-oxo-PGF_1α synthesis by bovine seminal vesicle (BSV) PG synthase was stimulated by the cytosolic fractions of each tissue in a dose-dependent manner. The cytosols from decidua vera and placenta were the most effective in stimulating synthesis and also stimulated PGE₂ biosynthesis. Reduced glutathione (GSH) acted to increase the biosynthesis of PGE₂ at the expense of other PGs both in the presence and absence of various cytosols. These data are indicative that the mode of action of cytosolic fractions on the stimulation of PG biosynthesis is unlike that of GSH. Indomethacin and aspirin, inhibitors of fatty acid cyclooxygenase activity, strongly inhibited the cytosol-induced stimulation of BSV PG synthase.The cytosolic factors that stimulated PG biosynthesis exhibited differential behavior towards boiling and dialysis. The stimulatory effect of all cytosolic fractions was sensitive to boiling except in the case of chorion leave effects toward 6-oxo-PGF_1α production. In dialysis studies we found that the cytosolic components that stimulated the production of PGF_2α were not removed by dialysis except in the case of cytosol of placenta whereas the stimulatory effects of various cytosols toward the biosynthesis of PGE₂ and 6-oxo-PGF_1α were removed by dialysis. These results are indicative of the presence of endogenous factors in human intrauterine tissues that preferentially stimulate the biosynthesis of PGF_2α and 6-oxo-PGF_1α and are further suggestive that PC biosynthesis in intrauterine tissues is, at least in part, regulated by cytosolic factors.     

The use of a potential-sensitive cyanine dye for studying ion-dependent electrogenic renal transport of organic solutes. Uptake of l-malate and d-malate by luminal-membrane vesicles

The mechanisms of uptake of dicarboxylic acids by rabbit renal luminal-membrane vesicles were studied by the use of filtration and spectrophotometric techniques as described in an accompanying paper [Kragh-Hansen, Jørgensen & Sheikh (1982) Biochem. J. 208, 359–368]. Addition of l- or d-malate to dye-membrane-vesicle suspensions in the presence of Na⁺ gradients (extravesicular>intravesicular) resulted in spectral curves indicative of depolarization events. The renal uptake of dicarboxylic acids was dependent on the type of Na⁺-salt anion present and could be correlated with the ability of the anions to penetrate biological membranes (i.e. Cl⁻>SO₄²⁻>gluconate). Identical results were obtained by a filtration technique with Sartorius membrane filters. The results indicate that the dicarboxylic acids are taken up by the membrane vesicles in an electrically positive form (i.e. Na⁺/substrate coupling ratio 3:1) by an Na⁺-dependent transport system. This proposal was further supported by spectrophotometric experiments with various ionophores such as valinomycin, gramicidin and nigericin. The absorbance changes associated with simultaneous addition of l- and d-malate and spectrophotometric competition studies revealed that the two isomers are taken up by a common transport system. Spectral changes of the dye induced by addition of increasing concentrations of l- or d-malate indicated that the transport system favours the unphysiological d-form rather than the l-form of malate. Furthermore, it was observed that the affinity of both isomers for the transport system was dependent on the concentration of Na⁺ in the medium.     

Identification of the insulin-like growth factor binding proteins 5 and 6 (IGFBP-5 and 6) in human breast cancer cells.

Four estrogen receptor-positive (ER+) [MCF-7, T47D, ZR75 and BT474] and 3 ER- [Hs578T, MDA-MB-468 and MDA-MB-231] human breast cancer cell lines were examined for expression of the IGFBP-5 and IGFBP-6 genes. Northern blot analysis revealed that all cell lines, except MDA-MB-231, expressed IGFBP-5 mRNA. IGFBP-6 mRNA, however, was expressed only by the ER- cell lines. Western immunoblotting indicated that the previously unidentified 31-kDa and 32-kDa IGF binding species secreted by these cell lines are IGFBP-5. The levels of IGFBP-4 and IGFBP-5 were increased in MCF-7 cells by estradiol and IGF-I, respectively, indicating that these BPs may contribute to the growth stimulatory response to these mitogens.     

Translation of preprochymosin in vitro. Evidence for folding of prochymosin to the native conformation.

The uptake and metabolism of 35S-labelled sulphur amino acids were compared in periportal (PP) and perivenous (PV) rat hepatocytes, isolated by digitonin/collagenase perfusion, to identify the factors underlying the previously observed [Kera, Penttilä & Lindros, Biochem. J. (1988) 254, 411-417] higher rate of GSH replenishment in PP cells. The buthionine sulphoximine-inhibitable synthesis of GSH was faster in PP than in PV hepatocytes with both cysteine (6.1 versus 5.0 mumol/h per g of cells) and methionine (4.5 versus 3.3 mumol/h per g) as well as with endogenous precursors and L-2-oxo-4-thiazolidinecarboxylate as substrates. However, the uptake of cysteine by PP cells was slower than by PV cells (8.6 versus 10.3 mumol/h per g of cells), whereas methionine was taken up at similar rates. The activity of gamma-glutamylcysteine synthetase (GCS) was slightly higher in digitonin lysates from the PP than from the PV zone. Production of sulphate, the major catabolite of [35S]cysteine sulphur, as well as incorporation of the label into protein occurred at similar rates in PP and PV cells. Taurine, on the other hand, was produced from [35S]cysteine much faster by PV than by PP cells (0.7 versus 0.1 mumol/h per g of cells). Accordingly, the taurine content of PV hepatocytes tended to be higher and to increase faster during incubation with methionine. These results imply that metabolism of taurine is highly zonated within the acinus. They also suggest that both the slightly lower GCS activity and the fast metabolism of cysteine to taurine limit the capacity of PV hepatocytes to synthesize GSH.     

A new selective medium for isolating Pseudomonas spp. from water.

A new medium, pseudomonas selective isolation agar, was developed to isolate Pseudomonas spp. from water. It consists of 350 micrograms of nitrofurantoin per ml and 2 micrograms of crystal violet per ml in a nutrient agar base. It is more selective for Pseudomonas spp. than are available commercial media. Its ingredients are inexpensive and readily available, and it is easy to prepare.     

Hybrid molecules containing the A-domain of insulin-like growth factor-I and the B-chain of insulin have increased mitogenic activity relative to insulin

Two synthetic insulin-like compounds consisting of the B-chain of insulin linked via disulfide bonds to A chains corresponding to the A-domain or the A- and D-domains of insulin-like growth factor I (IGF-I) have been evaluated for mitogenic activity and for binding to IGF receptors and IGF carrier proteins. Both compounds are 3- to 5-fold more potent mitogens than insulin, and have a comparably increased affinity for the type I IGF receptor that mediates these mitogenic effects in chick embryo fibroblasts. Neither compound interacts with IGF carrier proteins. These results indicate that the A-domain of IGF-I is importantly involved in its growth-promoting properties.     

Third form of calcium-activated neutral proteinase from calf brain: purification, partial characterization and comparison of properties with other forms

A third form (CANP3) of calcium-activated neutral proteinase (CANP) has been purified, 3900-fold, to near homogeneity from calf brain cortex. The purification procedure is based on the one recently developed for the purification of CANP1 and CANP2. The molecular weight of CANP3, as judged on SDS-polyacrylamide gel electrophoresis was Mr 78,000. A protein with an apparent Mr 17,000 co-purified with the proteinase. At neutral pH (7.2), it was maximally active at 260 microM CaCl2. In the presence of CaCl2, CANP1 and CANP3 were autolyzed very rapidly, whereas the autolysis of CANP2 was slow and gradual. The autolyzed CANP1 and CANP3 responded differently to CaCl2; CANP1 lost activity completely, whereas CANP3 was fully active at 0.5 microM CaCl2. Despite the opposite behavior of these proteinases in the presence of Ca2+, no significant differences in the peptide maps of the three proteinases were observed. Neurofilaments, neurotubules and myelin basic protein (MBP) were degraded by each of the proteinases. Monoclonal antibodies raised against CANP2 reacted almost equally with CANP1 and CANP3. As with CANP1 and CANP2, leupeptin and sulfhydryl-modifying compounds, NEM and iodoacetic acid, inhibited the activity of CANP3.     

Regulation of high molecular weight bovine brain neutral protease by phospholipids in vitro

The activity of the heat stable, glycosylated high molecular weight bovine brain neutral protease (HMW protease) is differentially regulated by phospholipids. While phosphatidylcholine (PC), phosphatidylserine (PS) and phosphatidic acid (PA) had only marginal stimulatory effect (40–75%) on the activity of HMW protease, lysophoshatidylcholine (lysoPC) and lysophosphatidic acid (lysoPA) activated the enzyme by more than two-fold. Both lysoPC and lysoPA exhibited concentration-dependent saturation kinetics for the activation of HMW protease. Surprisingly, phosphoinositides (phosphatidylinositol, PI; phosphatidylinositol 4-phosphate, PIP; and phosphatidylinositol 4,5-bisphosphate, PIP₂) modulated the activity of protease differently: activation of the enzyme was higher with PIP (90%) as compared to PI (21%), whereas PIP₂ inhibited the enzyme (16%). The inhibition of the protease by PIP₂ was concentration-dependent. During receptor-coupled cell activation, phospholipase A₂ (PLA₂) converts PC and PA to lysoPC and lysoPA, respectively; PI is converted to PIP₂ by successive enzymatic phosphorylation by PI 4-kinase and PIP 5-kinase; and phospholipase C (PLC) degrades PIP₂ to diacylglycerol and inositol 1,4,5-trisphosphate. Therefore, the data suggest that HMW protease may be coupled to cell signal transduction where PLA₂, PI 4-kinase, PIP 5-kinase and PLC are involved.     

Synthesis of cationic cardiolipin analogues

An approach was developed to synthesize a new class of cationic cardiolipin analogues containing two quaternary ammonium groups with tetra alkyl groups retaining "glycerol" moiety, the central core of the molecule. Cationic cardiolipin analogues were modified via introduction of either two or four oxyethylene groups to enhance the solubility in polar solvents. These newly synthesized cationic cardiolipin analogues can be applied to a broad range of drug delivery systems such as transfection reagents.     

Alteration in amino-glycerophospholipids levels in the plasma of children with autism: a potential biochemical diagnostic marker

Currently, there is no biochemical test to assist in the behavioral diagnosis of autism. We observed that levels of phosphatidylethanolamine (PE) were decreased while phosphatidylserine (PS) were increased in the erythrocyte membranes of children with autism as compared to their non-autistic developmentally normal siblings. A new method using Trinitrobenezene sulfonic acid (TNBS) for the quantification of PE and PS (amino-glycerophospholipids, i.e., AGP) in the plasma of children was developed and standardized. Wavelength scans of TNBS-PE and TNBS-PS complexes gave two peaks at 320 nm and 410 nm. When varying concentrations of PS and PE were used, a linear regression line was observed at 410 nm with TNBS. Using this assay, the levels of AGP were found to be significantly increased in the plasma of children with autism as compared to their non-autistic normal siblings. It is proposed that plasma AGP levels may function as a potential diagnostic marker for autism.