Immunological aspects of retinoids in humans. II. Retinoic acid enhances induction of hemolytic plaque-forming cells

The effects of retinoic acid (RA) on the induction of antibody-producing cells from human tonsillar lymphocytes sensitized to sheep erythrocytes (SRBC) have been evaluated. Our results indicated that 10(-5) to 10(-7) M RA caused up to a three-fold increase in the number of plaque-forming cells (PFC) and a qualitative increase in the size of the plaques during the induction of PFC in 5- to 7-day cultures. Enhancement also occurred when tonsil cells were preincubated with RA for 24 hr and then washed, or when RA was added any time in the first 4 days after initiation of the culture. When T- and B-cell fractions were pretreated with RA for 24 hr, washed, and recombined with SRBC, RA-induced augmentation of PFC occurred only in conjunction with RA treatment of the B-cell fraction. Pretreatment of the T-cell fraction had no effect on PFC induction or on the RA-enhanced response when the B-cell fraction was simultaneously treated with RA. Other experiments suggested that RA did not modulate PFC induction by influencing regulatory functions of adherent accessory cells. Our study demonstrates that RA can enhance human antibody responses and shows that this effect is not caused by increased activity of T cells or adherent accessory cells, but is instead the result of a direct effect of RA on B-cell populations.     

Studies on the mechanism of the human natural killer cell lethal hit: a new method for rapid physical isolation of lethally hit K562 targets and inhibition of cytolysis by reduced temperature or trypsin

A new method was developed which allows for rapid (2 min) physical isolation of viable K562 target cells after being programmed to lyse (lethally hit) by purified human natural killer (NK) cells (LGL). To achieve this K562 cells which were obtained from the 34-36% interface of discontinuous Percoll gradients and purified human NK cells (LGL) which were obtained from the (43-45% Percoll) interface were employed. Using a Ca2+ pulse method and the separation of NK-K562 conjugates with EDTA and rapid centrifugation on Percoll gradients at 4 degrees C we could physically isolate the lethally hit K562 cells from the LGL allowing the study of the events leading to their subsequent lysis. Lysis of "purified" lethally hit K562 cells occurred in the absence of Ca2+ or Mg2+ and was blocked by reduced temperature (4 degrees C), or by the protease enzyme trypsin. When lethally hit targets were held at 4 degrees C (to block lysis) then rewarmed to 37 degrees C lysis ensued but with a rate slower than that of control cells not held at 4 degrees C. These data support the concept that transfer of protease-sensitive and possibly temperature-dependent structures from the NK cell to the target is a requisite step in NK cytolysis.     

Regulation of acetylcholinesterase activity by retinoic acid in a human neuroblastoma cell line

The ability of retinoic acid (RA) to modulate acetylcholinesterase (AChE) activity in a human neuroblastoma cell line (LN-N-5) was examined. The specific activity of AChE was significantly increased 3 days after exposure of LA-N-5 to RA and reached its maximum values after 9 or more days of culturing. Dose-response experiments demonstrated that large increases of AChE occurred at RA concentrations between 10(-7) and 10(-6) M with maximum AChE values detected at 10(-6)-10(-5) M. Increased AChE activity paralleled neurite outgrowth in LA-N-5 cultures. These findings demonstrate that RA can regulate specific AChE activity in human neuroblastoma cells in a manner consistent with neuronal maturation.     

Estrogens before birth and development of sex-related reproductive traits in the female guinea pig

Influences of estrogens on the differentiation of psychosexual traits in the female guinea pig were studied. Pregnant animals were injected intramuscularly with either 1, 2, or 3.3 micrograms estradiol benzoate (EB) or with 1 or 3 micrograms diethylstilbestrol dipropionate (DESDP). Injections were started on the 29th day of pregnancy, given daily for 6 days, and continued every other day until parturition. Female offspring were evaluated for onset of puberty, ovarian function, and lordosis and mounting behavior in adulthood. Prenatal treatment with 3 micrograms DESDP caused delayed puberty, impaired ovarian function, reduced responsiveness of lordosis to EB and P in adulthood (defeminization), augmented mounting in the absence of hormones (masculinization), and reduced responsiveness of mounting to exogenous EB and P in adulthood (defeminization). Prenatal treatment with 1 microgram DESDP produced similar but less pronounced effects. Prenatal treatment with 3.3 micrograms EB also caused a delay in puberty. However, responsiveness of lordosis to EB and P in adulthood was enhanced by treatment with either 1 or 3.3 micrograms EB prenatally. Further, neither mounting in the absence of hormones nor mounting in response to EB and P in adulthood were affected in any measurable way by any prenatal treatment with EB. These results show that estrogens can have masculinizing and defeminizing effects on sexually dimorphic reproductive traits in guinea pigs. The failure of EB to duplicate or parallel the effects of DESDP is not completely understood at this time, but it may indicate that less of the active substance reaches the target tissues following maternal and placental metabolism of EB than of DESDP.     

Differential effects of positive and negative proliferative stimuli on murine cytolytic and helper T-cell clones

Studies were performed to determine whether substances could be identified which exhibited differential regulatory effects--either positive or negative--on the growth of murine alloreactive cytolytic (Tc) and helper (Th) cloned T-cell lines. The following lines of evidence suggested that Tc and Th proliferate in response to the same growth factor (GF). (1) When GF-containing fluids from cultures of phorbol myristic acetate (PMA)-activated EL4 thymoma were fractionated by a variety of biochemical techniques. Tc and Th eluted together. (2) Absorption of GF-containing supernatants with either cloned Tc or cloned Th depleted GF activity for each to a similar extent, and GF eluted from either Tc or Th to which it had adsorbed supported the proliferation of Tc and Th equally well. (3) Lectin-depleted supernatants from cultures of concanavalin A (Con A)-activated Th stimulated the proliferation of Th as well as Tc. (4) Recombinant human interleukin (IL-2) supported the growth of Tc and Th with equal efficiency. On the other hand, the following observations indicated that Tc and Th differed in their responses to inhibitors of GF-driven proliferation. (1) Con A at greater than or equal to 0.3 micrograms/ml inhibited the GF-driven proliferation of each of three Th lines but not either of two Tc lines. To the contrary, Con A enhanced GF-dependent proliferation of Tc. (2) Like Con A, allogeneic splenocytes selectively depressed GF-driven proliferation of Th but not Tc. (3) A substance generated during the acid elution of GF from cells, possibly a modified fetal calf serum component, greatly reduced the GF-driven proliferation of Tc but not Th. These results suggest that differential control of the proliferation of Tc and Th in cellular immune responses may be achieved via negative regulatory signals and raise the possibility that substances which can selectively depress the proliferation of specific T-cell subsets might be found which would be of therapeutic value.     

Natural killer cell activity correlates with the amount of beta 2-microglobulin on human peripheral blood lymphocytes

Two cytotoxic assays, lectin-dependent cytotoxicity and natural killer (NK) cytotoxicity, were used to assess the competence of cord blood and neonatal peripheral blood mononuclear cell (PBMC) and T-cell cytotoxic reactions. The effect of exogenous interferon was also studied. Results were compared with cytotoxic capabilities of adult cells and cells from patients with primary immunodeficiency syndromes. Lectin-dependent cytotoxicity (LDCC), a property of both T and non-T cells, was assessed by lysis of chromium-labeled EL4 tumor target cells in the presence or absence of exogenous fibroblast interferon (IFN-β). Natural killer cytotoxicity was assessed by lysis of two different chromium-labeled tumor target cells, Molt 4f and K562 in the presence or absence of IFN-β. Lectin-dependent cytotoxicity (LDCC) of PBMC of cord blood (32 ± 4% SEM) and adult cells (36 ± 2% SEM) were equivalent but neonatal cells had slightly decreased LDCC (22 ± 3% lysis). T-depleted cells from cord or neonatal blood had increased LDCC but T-enriched (>95% sheep erythrocyte rosette-forming cells) from both cord (22 ± 3%) and neonatal blood (18 ± 5%) had significantly reduced LDCC compared to 55 ± 2% for adult T cells. This deficiency corrects with age and is near normal after age 2. Preincubation with IFN-β did not enhance LDCC of newborn or adult cells. The LDCC of some cord T cells was markedly reduced and was in the same low range as patients with severe combined immunodeficiency. Natural killer (NK) cytotoxicity of PBMC from cord and adult cells was equivalent at three effector:target ratios against the Molt 4f target but against the K562 target, cord PBMC had significantly less NK activity (22 ± 11 SD) compared to adult NK activity (50.5 ± 22.2 SD) at a 50:1 effector:target ratio. Similar differences were noted at 25:1 and 10:1 target:effector ratios. NK cytotoxicity against Molt 4f targets of adult cells was significantly enhanced by preincubation with IFN-β but NK of cord cells was only variably enhanced. By contrast, IFN-β enhanced NK against K562 targets of both adult and cord cells, adult greater (67.7 ± 20) than cord cells (37.8 ± 2.0). These T-cell effector deficiencies are in marked contrast to the vigorous proliferative responses of newborn T cells, and parallel deficiencies of certain neonatal lymphokines. These defects may explain the newborns' enhanced susceptibility to intracellular viruses and to congenital viral infections.     

Regulation of NK cell activity by prostaglandin E2: the role of T cells

The influence of T cells on the production of prostaglandins (PGE2) and on PGE2-mediated regulation of natural killer (NK) activity was studied. Supernatants from peripheral blood mononuclear cells (PBMC) and from PBMC depleted of T cells ((PBMC)-T), both of which had been incubated in plastic petri dishes overnight, contained similar amounts of PGE2, as detected by radioimmunoassay and by their potential to inhibit NK activity of peripheral blood mononuclear cells in a 51Cr release assay with K 562 cells as the target population. However, the NK activity of PBMC was inhibited significantly more strongly (P less than 0.005) by PGE2-containing supernatants than was the NK activity of (PBMC)-T. In further assays, in which synthetic PGE2 in concentrations of 10(-4) and 10(-5)M was added, a significant inhibition of NK activity was observed in PBMC populations (P less than 0.05), but not in (PBMC)-T. Thus, T cells did not seem to be involved in the control of PGE2 production, but their presence was necessary for PGE2-mediated inhibition of NK activity.     

Membrane defects of the tolerant B cell. I. Failure of antigen-induced capping.

In order to study the membrane function of tolerant B antigen-binding cells, tolerance to the trinitrophenyl (TNP) determinant was induced in mice by injecting the reactive form of the hapten, trinitrobenzene sulfonic acid (TNBS). By appropriate transfer experiments, Fidler and Golub (J. Immunol.112, 1891, 1974) had previously shown that this form of tolerance is a B-cell property, induced and expressed in the absence of T cells. Hapten inhibition demonstrated the TNP-specificity of receptors on TNP-donkey erythrocyte(TNP-D)-binding cells in tolerant and nontolerant mice. About 88% of these cells were B cells by immunofluorescence, and the remainder were T cells. In the tolerant mice, challenge with TNP-sheep erythrocytes failed to expand the TNP-binding population, but sheep erythrocyte binders and anti-sheep plaque-forming cells expanded normally. Despite little or no change in TNP-binding cell numbers after tolerance induction, the TNP-binding cells of tolerant animals could not cap their receptors, in contrast to the sheep erythrocyte-binding cells from the same animals which capped normally. Although there is no anti-TNP plaque-forming cell response when tolerogen and immunogen are given simultaneously, capping failure is not evident until 2–4 days after tolerogen exposure. By Day 7, substantial recovery of immune responsiveness had occurred, yet even 12 months after a single dose of tolerogen there was no restoration of capping. Thus despite the association of both capping failure and unresponsiveness with tolerogen exposure, these lymphocyte functional defects appeared not to be causally related.     

Membrane defects in the tolerant B cell. II. Mechanism of capping failure and reversal by antigen exposure.

The demonstration that TNP-binding B lymphocytes from animals whose B cells have been rendered tolerant to TNP by trinitrobenzene sulfonic acid cannot undergo antigen-induced capping of their TNP receptors for at least a year despite recovery of immune responsiveness has led to a search for the mechanism of the capping failure. Microtubule-dependent membrane “locking” analogous to that induced by concanavalin A appears to afflict the tolerant B cells, in that capping TNP receptors is restored after exposure to 10^?4M colchicine or overnight incubation at 4 °C. Assignment of the defect to the cytoskeleton rather than the receptors themselves is also supported by the observations that enzymatic stripping and regrowth of receptors does not unlock the cell and that non-Ig membrane molecules recognized by antilymphocyte serum also cannot be capped on the tolerant cells. Cells which have remained locked for 4 days to 8 months after a single tolerogen exposure become unlocked 4 days after immunogen is given. Four days after immunogen, tolerogen fails to lock the membranes of TNP-binding cells. These results suggest that tolerogen contact interferes in a much broader range of functions in the TNP-binding cell than those which affect the immune response. Among these effects is a remarkably stable “locked” configuration of the cytoskeleton which is independent of immune responsiveness or receptor turnover, but which can be reversed by exposure to immunogen whether or not an immune response ensues.     

Effects of retinoic acid (RA) on the growth and phenotypic expression of several human neuroblastoma cell lines

It has been shown that retinoic acid (RA) can promote morphologic differentiation and inhibit the growth of a human neuroblastoma cell line, LA-N-1. The present study tests the histological generality of these phenomena by determining the effects of RA on seven other human neuroblastoma cell lines. Results show that RA strongly inhibited anchorage-dependent growth and induced morphologic alterations in six of seven of the cell lines. These alterations included morphologic differentiation as evidenced by formation of neurite extensions in four of the lines, cellular enlargement and vacuolization in one culture, and formation of large, flattened epithelial or fibroblastic-like cells in another culture. Although one cell line was relatively insensitive to the effects of RA in monolayer culture, all seven were strongly inhibited by RA in soft agar assays. Cellular RA-binding proteins were detected in 2/2 lines tested. These findings suggest that, as a histological group, human neuroblastoma cells are extremely sensitive to RA-induced growth inhibition and morphological alterations generally associated with reduced expression of the malignant phenotype of this type of cancer.     

Butanedione treatment reduces receptor binding of a lysosomal enzyme to cells and membranes

Treatment of the lysosomal enzyme, α-L-iduronidase, with 2,3 butanedione, an arginine modifying reagent, under conditions where enzyme activity was unaffected, reduced by 50% the internalization of the enzyme into cultured human fibroblasts. The lowered rate of internalization was a result of a reduced binding of the enzyme to cell surface receptors. The butanedione treatment of α-L-iduronidase caused a 90% reduction of binding when isolated fibroblast membranes were used as the source of receptor. This marked reduction of binding was also seen when membranes from a rat chondrosarcoma were examined. Although there is ample evidence that the receptor recognizes mannose 6-phosphate residues on the enzyme, the results suggest that other structural features, such as arginine moieties, may also be important in iduronidase binding.     

A separation chamber to sort cells and cell organelles by weak physical forces: III. Preparative electrophoresis of cells in stationary density gradients at low electric field strength

An apparatus was designed for preparative density gradient electrophoresis of mammalian cells. In a low conductivity isotonic Ficoll density gradient of 1.5 cm length, human erythrocytes treated with neuraminidase were separated from untreated erythrocytes at an electric field strength of approximately 2.7 v/cm. Within 5 min two bands of erythrocytes were visible. Electrophoretic separation was completed within 25 min. The fractionation is performed in a design consisting of three Perspex circular plates, bottom and top plates of which can be displaced simultaneously relative to the stationary middle plate by a worm-gear mechanism. The middle plate contains a cylindrical separation chamber of 50 cm² and 1.5 cm high. Top and bottom plates contain cones and flow deflectors for the undisturbed thin layering of cell suspensions and for introduction of the density gradient. Also present in top and bottom plates are electrode compartments containing a large platinum electrode and a cellophane membrane that isolates the separation chamber hydrodynamically but not electrically from the electrode compartment. The electrode compartments were flushed with electrophoresis buffer to remove products of electrophoresis as well as the (low) generated Joule heat.     

Activation and mechanism of action of suppressor macrophages

Intravenous administration of Corynebacterium parvum to alloimmunized mice activates splenic suppressor macrophages that effectively curtail primary and secondary generation of cytotoxic T lymphocytes (CTLs) in vitro. CTL generation was significantly inhibited in suppressed primary cultures by Day 3, the earliest time point that activity is first detected in control cultures. Suppressor macrophages had to be present during the first 24–48 hr of culture to effectively curtail the generation of CTLs. However, if suppressor macrophages were reactivated by 48-hr in vitro culture and then added to primary sensitizations that had been initiated 48 hr previously, they were capable of significant suppression. Suppressor cells produced a soluble factor that mediated the inhibition of CTL generation. The production or action of this factor could not be counteracted by indomethacin.     

Enhanced sensitivity of trisomy 21 monocytes to the maturation-inhibiting effect ot interferon

Cultured peripheral blood monocytes from subjects with trisomy 21 (Down Syndrome) demonstrated a 3.7-fold enhanced sensitivity to the maturation-inhibiting effect of leukocyte interferon. This increased sensitivity is considered to be the result of the presence of an increased concentration of the interferon receptor, which is controlled by the IfRec locus on human chromosome 21, on the surface of the trisomic monocytes. Since macrophages are important components of many immune processes and interferon is itself a product of and has regulatory functions in immune reactions, the enhanced sensitivity of trisomic monocytes to interferon may be a factor leading, paradoxically, to the greater susceptibility of trisomic individuals to viral and bacterial infections.     

Rapid kinetics of lysis in human natural cell-mediated cytotoxicity: Some implications

The entire lytic process of natural cell-mediated cytotoxicity against sensitive target cells can occur rapidly, within minutes. This was demonstrated by ⁵¹chromium release and in single-cell assays. At the cellular level, most of the target cell lysis occurred within 15–30 min after binding to effector cells. The enriched natural killer cell subpopulation of lymphocytes obtained by Percoll density gradient centrifugation (containing >70% large granular lymphocytes (LGL)) was the most rapidly lytic population by ⁵¹chromium release. However, in the single-cell assay, the rate of lysis of bound target cells was quite similar for the LGL-enriched effector subpopulation and the higher density subpopulation of effector cells recognized previously. Both the light and dense effector cells contained similar numbers of target binding cells. Therefore, that the light subpopulation effected lysis more rapidly and to a greater extent than the dense subpopulation suggested that the low-density effector cells probably recycled more rapidly than those of higher density. This was corroborated by the finding that when conjugates were formed at 29 °C for the single-cell assay, a significant number of dead unconjugated targets could be observed only on the slides made with the LGL-enriched effector cells but not on those made with dense effector cell. Lysis continued to increase in the chromium-release assay probably because of recycling, recruitment, and/or heterogeneity of the effector cells, and/or because of heterogeneity or delayed death of the target cells.     

Evidence that the collagen in the culture medium of Chinese hamster lung cells contains components related at the primary structural level to the alpha1(V) collagen chain

The collagenous protein synthesized by cultured Chinese hamster lung (CHL) cells and present in the culture medium has been isolated after limited pepsin digestion and differential salt precipitation. Molecular size analysis of this material indicates that the CHL cell medium collagen contains chains which exhibit an apparent molecular mass of approximately 85,000 Da. When chromatographed on CM-cellulose under denaturing conditions, the reduced and alkylated CHL cell medium collagen chains elute slightly after the human alpha1(I) chain but well before the pepsin-derived alpha1(V) chain, which is the constituent chain present in the CHL cell cellular matrix collagen. Analysis of the peptides derived by CNBr cleavage of the CHL medium collagen chains by chromatography on CM-cellulose reveals, however, that these chains contain peptides which correspond both in size and in chemical properties to those derived from the alpha1(V) collagen chain, but clearly lack two peptides (alpha1(V)-CB4 and alpha1(V)-CB5) which are normally present in pepsin-derived alpha1(V) chains. Furthermore, analysis of the CHL cell culture medium collagenous material obtained without pepsin digestion indicates the presence of collagenous chains that exhibit after reduction a molecular mass of approximately 160,000 Da, which is smaller than the proposed size of the pro alpha1(V) collagen chain. These results demonstrate that the collagenous protein present in the culture medium of CHL cells is directly related at the primary structural level to the alpha1(V) collagen chain, and it is postulated that this material represents the large fragment derived from a collagenase cleavage of the [pro alpha1(V)]3 molecules present in the cell layer. Furthermore, these results and previous reports indicate that the only identifiable genetic type of procollagen chain synthesized by this cloned cell line in culture corresponds to the pro alpha1(V) chain.     

Pigeon liver malic enzyme: Involvement of an arginyl residue at the binding site for malate and its analogs

Treatment of malic enzyme with arginine-specific reagents phenylglyoxal or 2,3-butanedione results in pseudo-first-order loss of oxidative decarboxylase activity. Inactivation by phenylglyoxal is completely prevented by saturating concentrations of NADP⁺, Mn²⁺, and substrate analog hydroxymalonate. Double log plots of pseudo-first-order rate constant versus concentration yield straight lines with identical slopes of unity for both reagents, suggesting that reaction of one molecule of reagent per active site is associated with activity loss. In parallel experiments, complete inactivation is accompanied by the incorporation of four [¹⁴C]phenylglyoxal molecules, and the loss of two arginyl residues per enzyme subunit, as determined by the colorimetric method of Yamasaki et al (R. B. Yamasaki, D. A. Shimer, and R. E. Feeney (1981) Anal. Biochem., 14, 220–226). These results confirm a 2:1 ratio for the reaction between phenylglyoxal and arginine (K. Takahashi (1968) J. Biol. Chem., 243, 6171–6179) and yield a stoichiometry of two arginine residues reacted per subunit for complete inactivation, of which one is essential for enzyme activity as determined by the statistical method of Tsou (C. L. Tsou (1962) Acta Biochim. Biophys. Sinica, 2, 203–211) and the Ray and Koshland analysis (W. J. Ray and D. E. Koshland (1961) J. Biol. Chem., 236, 1973–1979). Amino acid analysis of butanedione-modified enzyme also shows loss of arginyl residues, without significant decrease in other amino acids. Modification by phenylglyoxal does not significantly affect the affinity of this enzyme for NADPH. Binding of l-malate and its dicarboxylic acid analogs oxalate and tartronate is abolished upon modification, as is binding of the monocarboxylic acid α-hydroxybutyrate. The latter result indicates binding of the C-1 carboxyl group of the substrate to an arginyl residue on the enzyme.     

Inhibition of natural killer cell lysis by natural killer-inhibitory substance: mechanism of suppression

The mechanism of suppression of NK-mediated lysis by a soluble product of peritoneal cells (NK-IS, natural killer-inhibitory substance) was investigated. Pretreatment of effector cells resulted in depressed NK lysis while pretreatment of targets had no effect, indicating suppression is due to alterations in effector cell function rather than changes in target cells. NK-IS had no effect on the formation of conjugates between effectors and NK-susceptible targets. When NK-IS was added to effector-target cell mixtures after the binding step had been successfully completed, ensuing lysis was significantly depressed, confirming that NK-IS inhibited a postbinding lytic event. The degree of suppression caused by NK-IS was directly related to the duration of exposure to the inhibitory molecule. In addition, a preliminary temperature-dependent step of binding to and/or intracellular entry of NK-IS into effectors is required before suppression can occur. NK-IS prevents the activation of NK cell lysis by interferon and Corynebacterium parvum and effectively inhibits lysis mediated by already activated effectors. The potent suppression of NK lysis and prevention of interferon and C. parvum-mediated activation of NK lysis by a soluble product of peritoneal cells may explain the extremely low level of NK effector cell function within the peritoneal cavity.     

Transport of methotrexate in Chinese hamster ovary cells: a mutant defective in methotrexate uptake and cell binding

A regulatory role for cytoplasmically derived proteins in chloroplast translation in organello was examined by analyzing protein synthesis in plastids isolated from cells of Euglena gracilis which had been treated with cycloheximide (CHI). Incorporation of [35S]methionine by chloroplasts from CHI-inhibited Euglena was reduced approximately 40 and 90% by exposure of the cells to the antibiotic for 2 and 4 h, respectively. The chloroplast translation products were then analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. The synthesis of polypeptides in the soluble compartment of the plastid was substantially diminished by as little as 15 min of CHI pretreatment. No qualitative alterations of the polypeptide pattern were detected. Qualitative changes were seen in the thylakoid fraction, however. Comparison of the stainable polypeptides and fluorographs of thylakoid membranes from CHI-treated cells with those of controls showed several instances in which the more slowly migrating member of a doublet accumulated with a concomitant depletion of a more rapidly migrating component. A pair of polypeptides at 28 and 30 kDa, which we believe are the Euglena homologs of the photogene product and its precursor, respectively, are representative of this phenomenon. Additionally, thylakoids from cells pretreated with CHI sometimes synthesized novel polypeptides larger than 65 kDa. Finally, when intact chloroplasts from CHI-inhibited Euglena were incubated with a postchloroplast supernatant from normal cells, there was a partial reversion of the anomalies seen in the fluorographs. These data are interpreted to indicate the cytoplasmic origin of one or more proteins whose function is to process chloroplast translation products.     

NADP-linked 15-hydroxyprostaglandin dehydrogenase from human placenta: partial purification and characterization of the enzyme and identification of an inhibitor in placental tissue.

An NADP-linked 15-hydroxyprostaglandin dehydrogenase has been identified in human placental tissue and partially purified. Prostaglandins of the A and B series are good substrates for this enzyme while those of the E and F series are not. This enzymic preparation also catalyzes oxido-reductions at the 9 position of the prostaglandin molecule; these are slow compared to those occurring at the 15 position of the prostaglandins in the A and B series. Disc gel electrophoresis of the purified enzyme reveals the presence of three protein bands which contain dehydrogenase activity. Boiled placental homogenates contain an inhibitor which appears to be specific for the NADP-linked 15-hydroxyprostaglandin dehydrogenase. The inhibitor is heat stable and has a molecular weight of 6,000 – 7,000.