Phospholipid turnover during cell-cycle traverse in synchronous Chinese-hamster ovary cells. Mitogenesis without phosphoinositide breakdown.

The turnover of phospholipids was investigated in quiescent serum-starved Chinese-hamster ovary (CHO-K1) cells stimulated to progress through the cell cycle by the addition of dialysed bovine serum. A variety of radiolabelling techniques were employed to study the rapid effects of serum on phospholipids and later events during G1 and S phases of the cell cycle. Pulse-labelling studies using [32P]Pi revealed that there was a stimulation of the synthesis rate of all phospholipids investigated during the initial few hours after serum addition. The greatest stimulation (20-fold) was observed in phosphatidylcholine, and the smallest in the polyphosphoinositides (PPIs). Mock stimulation with serum-free medium caused a similar increase in PPI turnover, but little or no effect on turnover of other phospholipids. This effect could be accounted for by a stimulation of the turnover of cellular ATP pools increasing [32P]ATP specific radioactivity. Late G1 and S phases were associated with a decrease in the rate of synthesis of all phospholipids. Phosphatidic acid was the only phospholipid whose labelling fell below that in mock-stimulated cells during the period of the cell cycle. Stimulation of serum-starved cells that had been prelabelled with myo-[2-3H]inositol caused no change in the amounts of inositol trisphosphate, but both serum-stimulated and mock-stimulated cells exhibited similar small decreases in both inositol bisphosphate and inositol monophosphate, of approx. 30% after 30 s. When cells were serum-stimulated in the presence of 10 mM-Li+, there was no increase in the size of the total inositol phosphate pool. We conclude that mitogenic stimulation and cell-cycle traverse cause profound and complex effects on phospholipid turnover in CHO-K1 cells, but there is no evidence for a role of inositol lipid turnover in the proliferative response to serum in this cell line.     

Trichome-borne and artificially applied acylsugars of wild tomato deter feeding and oviposition of the leafminer Liriomyza trifolii

Oviposition and adult feeding of the leafminer Liriomyza trifollii (Burgess) (Diptera, Agromyzidae) on Lycopersicon pennellii (Corr.) D'Arcy and its F₁ hybrid with Lycopersicon esculentum (Mill.) was significantly less than that on the cultivated tomato, L. esculentum. The resistance of L. pennellii and the F₁ was reduced following rinsing of foliage with ethanol. Resistant attributes of L. pennellii were transferred to L. esculentum through appression of L. pennellii foliage to L. esculentum leaflets. Application of purified 2,3,4-tri-O-acylglucoses (the principal component of type IV glandular trichome exudate of L. pennellii) to L. esculentum significantly decreased feeding and oviposition on L. esculentum leaflets by 61–99%. Therefore the principal mechanism of resistance to this leafminer by L. pennellii is the secretion of these acylglucoses. Dose response analysis of acylglucoses applied to L. esculentum shows that dosages as low as 10% those found on L. pennellii provide large reductions (91%) in leaf punctures and mines.     

Muscarinic release of inositol trisphosphate without mobilization of calcium in bovine adrenal chromaffin cells

Chromaffin cells of bovine adrenal medulla release catecholamines in response to activation of nicotinic ACh receptors which open voltage-sensitive calcium channels. Catecholamine secretion by exocytosis requires an increase in cytosolic free calcium. The cells also possess muscarinic ACh receptors but muscarinic agents do not provoke catecholamine release. Quin-2 studies show that they do not increase cytosolic free Ca2+ concentration, but unlike the nicotinic agents, they cause phosphoinositide hydrolysis. Muscarinic stimulation leads to rapid loss of labelled phosphatidylinositol 4-phosphate and of phosphatidylinositol 4,5-bisphosphate. At the same time there is release of inositol trisphosphate, inositol bisphosphate and inositol phosphate. In a number of other cells inositol trisphosphate may act as a second messenger releasing Ca2+ from storage sites in the endoplasmic reticulum but this is not its function in bovine chromaffin cells.     

ENZYMES OF PHOSPHOINOSITIDE METABOLISM DURING RAT BRAIN DEVELOPMENT

—The activities of four enzymes concerned with inositol lipid metabolism have been determined in homogenates of rat brains of different ages. The enzymes are CDP-diglyceride inositol phosphatidate transferase, phosphatidylinositol kinase, diphosphoinositide kinase and triphosphoinositide phosphomonoesterase. The activities of all the enzymes increased with age. Phosphatidylinositol kinase activity rose most sharply well before myelination, reaching a maximum at about 6 days of age. Diphosphoinositide kinase and triphosphoinositide phosphomonoesterase activities increased most rapidly during myelination. The increase in CDP-diglyceride inositol phosphatidate transferase showed no definite association with any period of development. It is concluded that triphosphoinositide metabolism is associated with myelin or a closely related structure.     

Variation in heat shock proteins within tropical and desert species of poeciliid fishes

The 70-kilodalton heat shock protein (hsp70) family of molecular chaperones, which contains both stress-inducible and normally abundant constitutive members, is highly conserved across distantly related taxa. Analysis of this protein family in individuals from an outbred population of tropical topminnows, Poeciliopsis gracilis, showed that while constitutive hsp70 family members showed no variation in protein isoforms, inducibly synthesized hsp70 was polymorphic. Several species of Poeciliopsis adapted to desert environments exhibited lower levels of inducible hsp70 polymorphism than the tropical species, but constitutive forms were identical to those in P. gracilis, as they were in the confamilial species Gambusia affinis. These differences suggest that inducible and constitutive members of this family are under different evolutionary constraints and may indicate differences in their function within the cell. Also, northern desert species of Poeciliopsis synthesize a subset of the inducible hsp70 isoforms seen in tropical species. This distribution supports the theory that ancestral tropical fish migrated northward and colonized desert streams; the subsequent decrease in variation of inducible hsp70 may have been due to genetic drift or a consequence of adaptation to the desert environment. Higher levels of variability were found when the 30- kilodalton heat shock protein (hsp30) family was analyzed within different strains of two desert species of Poeciliopsis and also in wild-caught individuals of Gambusia affinis. In both cases the distribution of hsp30 isoform diversity was similar to that seen previously with allozyme polymorphisms.      

Changes in [3H]inositol-labelled phosphoinositides of pig platelets in response to thrombin

Pig platelet phosphoinositides have been labelled with [³H]inositol and then treated with thrombin in the absence of Ca²⁺. There was a loss of labelled phosphatidylinositol 4,5-bisphosphate between 30 and 60 s after the addition of thrombin but the general picture was of increased labelling over a 4-min period. Labelling of phosphatidylinositol 4-phosphate showed no period of loss but there was an early loss of phosphatidylinositol and no increased labelling during the 4-min incubation. The small amount of lysophosphatidyl[³H]inositol in the platelets was not affected by thrombin treatment. Thrombin caused loss of [¹⁴C]arachidonate-labelled phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol.     

Carboranyl peptide-antibody conjugates for neutron-capture therapy: preparation, characterization, and in vivo evaluation.

Two model peptides rich in boron and prepared by Merrifield syntheses, dansyl.(nido-CB)2, (1) and dansyl.(nido-CB)10.Lys.Ac (2), where nido-CB represents the alpha-amino acid [nido-7-CH3-8-(CH2)3CH-(NH2)COOH-7,8-C2B9H10]-, were conjugated with the anti-CEA mAb T84.66 using peptide active ester reagents. The dansyl groups provided a means of fluorimetric analysis of mAb conjugates which was augmented by conventional amino acid analyses for nido-CB. The conjugate of 1 contained an average of 63 B atoms per mAb molecule. The mAb conjugate of 2 was chromatographically separated into a strongly fluorescent high molecular weight aggregated fraction (HMW) and a less intensely fluorescent monomeric fraction. Both fractions retained immunoreactivity. The HMW species contained an average of ca. 490 B atoms/mAb molecule, as determined by amino acid analysis. Biodistribution data were collected using nude mice bearing LS174T xenografts and 125I-labeled mAb conjugates. While the lightly B-loaded dipeptide conjugate gave biodistribution results which resembled those of native T84.66 mAb, the undecapeptide conjugate displayed greatly enhanced liver uptake and decreased tumor accretion. These results suggest that as the boron-containing burden on the supporting immunoprotein is greatly increased, as in the case of the T84.66-2 conjugate, loss of circulating conjugate to liver effectively competes with the desired tumor localization. Means which might be taken to circumvent this difficulty have been described elsewhere (ref 15).     

Brushing cytology in biliary tract obstruction

During a period of eight years (1980 to 1987), cytologic samples were obtained by brushing and reverse screw devices from 54 patients undergoing transhepatic cholangiography for evaluation of obstructive jaundice. Eight patients were excluded from this study, seven for inadequate follow-up and one because of unsatisfactory cytologic material. Of the remaining cases, 32 were cytologically diagnosed as adenocarcinomas; all but one patient proved to have malignant disease by histologic examination and/or through clinical follow-up. These included 21 pancreatic carcinomas, 6 bile duct carcinomas, 1 ampullary carcinoma, 1 gallbladder carcinoma and 2 metastatic carcinomas. In one case, the diagnosis of malignancy was found to be in error upon review of the cytologic smears. Of 14 patients with negative cytologic diagnoses, 7 were found to have malignant neoplasms and 7 had benign diseases. These findings indicate that, while a positive cytologic diagnosis is a reliable indicator of a malignant biliary obstruction, a negative result does not exclude malignancy.