Jean Deb Butler

Uteroglobin inhibits phospholipase A2 activity.

Although progesterone is known to produce quiescence in the mammalian uterus, the mechanism of this effect is not clearly understood. Here, we report that uteroglobin, a progesterone-induced small molecular weight (16K) protein, inhibits phospholipase A2(PLA2) derived from porcine pancreas as well as from the RAW 264.7 macrophage cell line. We speculate that progesterone may exert its antimotility effects on the uterus via uteroglobin which, by inhibiting PLA2, decreases arachidonic acid release and subsequently reduces prostaglandin levels in this organ. This may explain why progesterone is so vital for the maintenance of pregnancy in almost all mammals.

Lysosomal ceroid depletion by drugs: therapeutic implications for a hereditary neurodegenerative disease of childhood.

Neuronal ceroid lipofuscinoses (NCLs) are the most common hereditary neurodegenerative diseases of childhood. The infantile form, INCL, is caused by lysosomal palmitoyl-protein thioesterase (PPT) deficiency, which impairs the cleavage of thioester linkages in palmitoylated proteins, preventing their hydrolysis by lysosomal proteinases. Consequent accumulation of these lipid-modified proteins (constituents of ceroid) in lysosomes leads to INCL. Because thioester linkages are susceptible to nucleophilic attack, drugs with this property may have therapeutic potential for INCL. We report here that two such drugs, phosphocysteamine and N-acetylcysteine, disrupt thioester linkages in a model thioester compound, [14C]palmitoyl∼CoA. Most importantly, in lymphoblasts derived from INCL patients, phosphocysteamine, a known lysosomotrophic drug, mediates the depletion of lysosomal ceroids, prevents their re-accumulation and inhibits apoptosis. Our results define a novel pharmacological approach to lysosomal ceroid depletion and raise the possibility that nucleophilic drugs such as phosphocysteamine hold therapeutic potential for INCL.

Inhibition of thrombin-induced platelet aggregation by uteroglobin

Uteroglobin, a steroid-dependent, small molecular weight (15K) protein in the rabbit, inhibited thrombin-induced aggregation of both rabbit and human gel-filtered platelets (GFP). GFP aggregation by arachidonic acid was not affected by uteroglobin. There were no effects of uteroglobin on thrombin-induced clotting of plasma or purified fibrinogen, or inhibition of thrombin by antithrombin III. Additionally, preliminary results suggest that uteroglobin does not interfere with binding of thrombin to platelets. We suggest that inhibition of platelet aggregation by uteroglobin may function in preventing thrombosis and ensuring free flow of blood through the microvasculature of the uterus and the placenta and may induce some of the antimotility effects of progesterone on the uterus.

Pantetheinase Activity and Cysteamine Content in Cystinotic and Normal Fibroblasts and Leukocytes

Summary: Cysteamine is the most effective agent known for the reduction of the elevated cystine content of cells from patients with cystinosis. A defect in endogenous cysteamine generation could account for many of the metabolic features of this disorder. To test this hypothesis, we have developed improved methods for measuring pantetheinase (cysteamine-generating) activity and intracellular cysteamine levels and used these methods to measure such parameters in cystinotic and normal leukocytes and cultured skin fibroblasts. Pantetheinase activity as defined in the text was similar in extracts of cystinotic and normal cells [leucocytes, normal, 78 ± 15 (S.E.), cystinotic, 56 ± 6.4; fibroblasts, normal, 9.4 ± 1.5; cystinotic, 7.7 ± 1.7]. Cysteamine levels were normal in leukocytes from cystinotics receiving no cysteamine or doses of oral cysteamine too low to reduce leukocyte cystine content. The results indicate that the cause of cystinosis is unlikely to be related to a failure to generate or sustain normal intracellular cysteamine levels.Speculation: cystearnine is an extremely effective cystine depleting agent for cystinotic fibroblasts in vitro and can greatly reduce cystinotic leukocyte cystine content in vivo. Its pharmacologic properties suggest that it might prove to be of value in the therapy of cystinosis. However, we do not believe that a defect in endogenous cysteamine generation is a characteristic of cystinotic cells. The eventual elucidation of the cystinotic defect may require analysis of the permeability characteristics of cystinotic lysosomes or the discovery of presently unidentified pathways for lysosomal metabolism of cystine.

Cystic fibrosis and phosphatidylcholine biosynthesis

The cystic fibrosis (CF) gene defect may be associated with a defect in membrane recycling. We have investigated the metabolism of the main constituent of plasma membrane, phosphatidylcholine (PC). In this study of platelets and fibroblasts, we show an increased uptake of choline into PC of CF cells as compared with normal cells. No accumulation of PC was seen. Other patients with respiratory disease (not CF) showed normal rates of incorporation of choline into platelet PC. Platelets from heterozygote individuals showed intermediate turnover rates of choline incorporation into PC. The increase in choline incorporation into PC in CF platelets was not due to modified or increased sensitivity to either cAMP or prostaglandin E2. The total amount and the proportions of the major phospholipids in platelets of control and CF individuals were identical. These findings indicate an increased turnover rate of this phospholipid in CF cells rather than an increased net synthesis.

Lipochromosome mediated gene transfer: Identification and probable specificity of localization of human chromosomal material and stability of the transferents

SummaryUsing lipochromosomes (phospholipid-entrapped chromosomes) we have transferred the human HGPRT gene into HGPRT deficient mouse cells (A9) with a frequency of approximately 1×10−5 (Mukherjee et al., Proc. Natl. Acad. Sci. USA 75: 1361–1365; 1978). Two other genes located on the long arm of the human X-chromosome were also expressed in two independently derived populations of transferents (A9/GT3 and A9/GT4). We report here the chromosomal and enzymatic composition of human HGPRT-positive clones from each subpopulation analyzed in detail with alkaline Giemsa-11 staining.All the clones expressed human PGK and HGPRT, but one (A9/GT4C6) lacked human G6PD. In each of four clones examined microscopically, a small piece of presumptive human chromatin was visible in the karyotypes of most cells. The chromatin fragment was free or attached in each cell of an individual clone. When integrated, the human chromosomal fragment in each clone appeared associated with the centromere of the same telocentric A9 chromosome (No. 6 by Q-banding).These data suggest that: (a) substantial human chromosomal fragments can be transferred into recipient cells using the lipochromosome technique; (b) clones from human HGPRT positive A9 transferent subpopulations may or may not possess other human X-linked markers; (c) the stability of lipochromosomally transferred genes varied from clone to clone and stability is generally poor in the absence of continuous selection pressure (e.g., HAT); (d) when multiple X-linked human genes were transferred to mouse cells a cytologically detectable human chromosomal fragment was identified free or attached to a host chromosome; and (e) integration of transferred human chromosomal material into mouse chromosomes may occur at preferential site(s) in the recipient genome.

Cystinotic and normal fibroblasts: Differential susceptibility to cysteine toxicity in vitro

SummaryExtracellular cysteine concentrations between 0.5 and 2.5 mM resulted in death of normal but not cystinotic cells grown in Eagles minimal essential medium containing supplemental fetal bovine serum and antibiotics. Differential cell survival was determined by viable cell counting using Trypan Blue dye exclusion. In cocultivation experiments of [3H]thymidine-labelled cystinotic fibroblasts with nonradioactive normal fibroblasts, autoradiography confirmed the selective survival of cystinotic cells in medium containing 1 mM cysteine. At this concentration of 1 mM cysteine, intracellular cystine content increased slightly in surviving normal cells but not in cystinotic cells, which normally contain a high level of intracellular cystine. This comparative resistance of cystinotic fibroblasts to elevated extracellular cysteine concentrations forms the basis for an in vitro selective system for these mutant human cells. Further exploration of this resistance phenomenon may well expand the understanding of the molecular defect in cystinotic cells.

285 INHIBITION OF THROMBIN MEDIATED PLATELET AGGREGATION BY UTEROGLOBIN

One of the putative functions of pregnancy-specific proteins is the inhibition of platelet aggregation. This function may facilitate the free flow of blood through the uterine microvas-culature during pregnancy. Uteroglobin (UG) is a small molecular weight (15K) protein first discovered in the rabbit uterus during early pregnancy. The present investigation was undertaken to determine the effect of UG on platelet aggregation. Gel-filtered platelets (GFP) or platelet-rich plasma (PRP) from human volunteers and/or from rabbits were tested in an aggregometer with various inducers of aggregation including thrombin, ADP, collagen and arachidonic acid. Similar aggregation trials were carried out after preincubatlng GFP or PRP with various concentrations of UG.These results suggest that uteroglobin specifically prevents throrabin mediated platelet aggregation in both rabbits and humans. This effect may be due to phospholipase A2 inhibition.

564 TREATMENT OF GLUTATHIONE SYNTHETASE DEFICIENT FIBROBLASTS BY INHIBITION OF |[gamma]|-GLUTAMYL TRANSPEPTIDASE WITH SERINE-BORATE

Glutathione synthetase (GSH-S) deficiency (5-oxoprolinuria) results in decreased cellular glutathione (GSH) content (10-20% of normal), and secondary over-production of 5-oxoproline. γ-glutamyl transpeptidase (GGTP) is the primary catabolic enzyme for GSH, and inhibition of this enzyme might thus be an approach to correcting the consequences of GSH-S deficiency. L-serine inhibits fibroblast GGTP. Inhibition is markedly enhanced by sodium borate buffer, 20 mM serine-20 mM borate causing>95% inhibition. Serine-borate added to Eagles MEM produced a dose and time dependent increase in GSH content of GSH-S deficient cultured fibroblasts. GSH content was doubled at 24 hours with 40 mM serine-40 mM borate. Borate alone was without effect. Conversion of 14C-glutamic acid to 5-oxoproline by GSH-S deficient cells was decreased by 70% to near normal levels by 24-hour pre-treatment with 40 mM serine-borate. The increased cell GSH content may block overproduction of 5-oxoproline from excess γ-glutamylcysteine by feed-back inhibiting γ-glutamylcysteine synthetase. Treatment produced no apparent toxicity; cell amino acid concentrations were unaffected other than an increase in serine and phosphoserine. The study demonstrates the possible therapeutic value of an inhibitor of a major catabolic enzyme for a substrate decreased secondary to a deficiency of its synthetic enzyme.

873 SPONTANEOUS TRANSFORMATION OF CULTURED SKIN FIBROBLASTS INTO OSTEOBLASTS IN A CASE OF DYSPLASTIC INTRA DERMAL CALCIFICATION

A unique case of spontaneous dysplastic intradermal calcification was identified in a 14 month old child. She presented with a rash at birth which evolved to generalized calcified nodules and plaques. Extensive investigation ruled out known causes of dystrophic calcification. Skin biopsy revealed osteoblast-like cells (OB) surrounding an osteoid layer at the surface of dysplastic woven bone containing osteocytes (OC). Electron microscopy (EM) showed that the OB more closely resembled fibroblasts and the OC appeared to be non-functional. Alkaline phosphatase (AP) was identified by EM techniques in the outer surface of the fibroblast cell membrane 5 microns from the mineralizing front of fibrous bone and along collagen fibers. X-ray microanalysis of the woven bone revealed mineral composition (Ca 41%; P 19%) similar to normal bone but less dense. Skin fibroblasts in vitro apparently transformed to OB after the 4th passage as evidenced by positive Von Kossa staining, high AP activity (201 units/mg protein with 70% heat labile), and morphological appearance. Cyclic-AMP phosphodiesterase activity was markedly elevated in the patients fibroblasts (mean 708 p moles/mg/min) compared to controls (mean 212 pmole/mg/min). This patient may provide a unique model for studying OB differentiation in vivo and in vitro.