Schistosomin, an antagonist of calfluxin

The percentage of Ca2+ positive mitochondria in the cells of the albumen gland of Lymnaea stagnalis was used as a measure for the effect of the gonadotropic hormone calfluxin (CaFl) and for the interaction between schistosomin, an agent present in the hemolymph of snails infected with the schistosome Trichobilharzia ocellata, and CaFl. From 3 weeks postexposure on glands of infected snails showed a lower response to CaFl than glands of noninfected snails. The response decreased with infection time. Apparently the glands are already affected at an early stage of parasitosis. Albumen glands of noninfected snails preincubated in serum of infected snails and subsequently incubated with CaFl gave a lower response to CaFl than control glands. The response of preincubated glands appeared to increase when the glands were rinsed in Ringer during increasing periods of time (up to 1 hr) before being incubated with CaFl. This indicates that schistosomin binds relatively tightly to albumen glands, probably to a receptor complex. Rinsing (1-2 hr) did not influence the (low) response to CaFl of glands of infected snails, whereas the response of glands of noninfected snails increased considerably after a 1- or 2-hr period of rinsing. The results probably indicate that only a low number of receptors for CaFl is still present in glands of infected snails. In glands exposed first to schistosomin and then to CaFl, the inhibition of the effect of CaFl was stronger than in glands exposed to schistosomin and CaFl at the same time. The response to CaFl appeared to increase when a higher concentration of CaFl was used. These data indicate that schistosomin and CaFl interact at the level of receptor complex(es) in the gland. It is argued that both substances not necessarily act at the level of one and the same receptor complex.     

Trichobilharzia ocellata: interference with endocrine control of female reproduction of Lymnaea stagnalis

Calfluxin (CaFl), one of the gonadotropic hormones of Lymnaea stagnalis, stimulates the influx of Ca2+ into the mitochondria of the cells of the albumen gland, one of the accessory sex organs of the snail. This effect is suppressed in glands of noninfected snails by an agent (schistosomin) present in the hemolymph of snails infected by Trichobilharzia ocellata as shown in in vitro experiments. The agent is present from 6 weeks postinfection onward. Ca2+ deposits in the mitochondria were demonstrated with the ultracytochemical antimonate precipitation technique. The percentage of Ca2+-positive mitochondria was taken as a measure for the effects of CaFl. This percentage appeared to be greatly reduced when glands were incubated in serum of infected snails (Sinf). The data showed that Ringer incubations can serve as controls for experiments with serum: no differences were found between Ringer incubations and incubations in either fresh or frozen serum of noninfected snails. Schistosomin was not affected by freezing, which enables cold storage of Sinf. The dose-response relationship of schistosomin shows that at a 1:2 dilution of Sinf with Ringer the response to CaFl was reduced more than 50%. Schistosomin is heat-stable and Pronase-labile, which indicates that it has a peptide nature. Probably schistosomin(s) is responsible for the reduction/cessation of fecundity in trematode-infected snails.     

Neuropeptide schistosomin inhibits hormonally-induced ovulation in the freshwater snail Lymnaea stagnalis.

This study examines the interaction between the caudodorsal cell hormone (CDCH) and schistosomin, a peptide secreted by the central nervous system of the snail (Lymnaea stagnalis) infected with the avian schistosome Trichobilharzia ocellata. Non-infected snails were injected with synthetic as well as native CDCH in the absence or presence of purified schistosomin. The response to 2 pmol of synthetic CDCH was blocked for 90% by coinjection with 3.5 pmol of schistosomin. The ovulation-inducing activity of extracts of cerebral commissures (the storage area of native CDCH) was also blocked by schistosomin. The degree of inhibition (65%), however, was less than that observed with synthetic CDCH. These results show that schistosomin inhibits ovulation and egg laying in Lymnaea. This explains the decrease or absence of egg laying in schistosome-infected freshwater snails.     

Phagocytic activity of hemocytes of M-line Biomphalaria glabrata snails: effect of exposure to the trematode Echinostoma paraensei

The phagocytic activity of hemocytes from 6-8-mm M-line Biomphalaria glabrata snails was studied in an in vitro assay using glutaraldehyde-fixed sheep erythrocytes (SRBC) as target cells. For individual snails, the percentage of hemocytes ingesting SRBC during a 1-hr interval, termed the phagocytic activity index (PAI), was determined. Hemocytes from snails infected for 1 day with Echinostoma paraensei had a slightly elevated PAI, but at both 8 and 30 days postexposure (DPE), hemocytes from infected snails had a significantly lower PAI than controls. Hemocytes taken from snails at 8 DPE also had a low PAI using rabbit erythrocytes and yeast as target cells. The low PAI at 8 DPE is attributed to the presence of large numbers of poorly spreading hemocytes with low phagocytic activity. Hemocytes from snails with 30-day infections were well spread but nonetheless had a low PAI. The presence of plasma from 8-day infected snails did not alter the PAI of hemocytes from control snails, nor was the PAI of hemocytes from infected snails changed by plasma from control snails. SRBC preincubated for 60 min in plasma from various groups of M-line snails did not elicit an increase in PAI when presented to hemocytes from control snails; in some cases, as with plasma from 6-8-mm control snails, such preincubation significantly reduced the PAI below levels obtained using SRBC preincubated in culture medium. As compared to hemocytes from snails with normally developing, 8-day-old intraventricular sporocysts (IS), hemocytes from snails exposed to infection but subsequently lacking IS had a significantly higher PAI.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adenylyl cyclase system of the small preovulatory follicles of the domestic hen: responsiveness to follicle-stimulating hormone and luteinizing hormone

The purpose of this study was to determine if the granulosa cells of the small preovulatory follicles of the domestic hen are a target tissue for follicle-stimulating hormone (FSH). The third largest (F3), fourth largest (F4), and fifth largest (F5) follicles were removed from hens at 20, 12, 6 and 2 h before ovulation of the F1 follicle. Basal, FSH- and luteinizing hormone (LH)-stimulable adenylyl cyclase (AC) activities were measured in the granulosa cells. Isolated granulosa cells of the F5 follicle, obtained 20 h before ovulation of the F1 follicle, were incubated with ovine (o) or turkey (t) FSH and progesterone (P4) was assayed in the medium. Basal AC activity was similar for F5, F4 and F3 granulosa cells except for an increase (P less than 0.01) in F3 follicles removed 2 h before ovulation of the F1 follicle. The FSH-stimulable AC activity of F5, F4 and F3 granulosa cells was elevated over basal (P less than 0.01). The greatest responsiveness was seen in the F5 follicle and the least in the F3 follicle. LH-stimulable AC activity was absent in the F5 follicle but present in the F4 and F3 follicles with the greater responsiveness in the F3 follicle. Isolated F5 granulosa cells secreted significant amounts of P4 in response to oFSH and tFSH. The data indicate that: 1) FSH stimulates the AC system of granulosa cells of the smaller preovulatory follicles (F5 greater than F4 greater than F3) while LH stimulates the AC system of granulosa cells of the larger follicles (F3 greater than F4), and 2) FSH promotes P4 production by granulosa cells of F5 follicles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation of schistosomin, a neuropeptide which antagonizes gonadotropic hormones in a freshwater snail

The molecular mechanisms underlying parasite-induced inhibitory effects on host reproduction were studied in the freshwater snail, Lymnaea stagnalis, infected with the schistosome parasite Trichobilharzia ocellata. This combination is used as a model system for host-parasite interactions involved in schistosomiasis transmission. The female gonadotropic snail neuropeptide, calfluxin, was labelled with fluorescein isothiocyanate (FITC) and used as a ligand in receptor-binding studies on membranes of its target organ, the albumen gland. The binding of calfluxin to its receptor-guanyl-nucleotide-binding-protein (G-protein) complex was inhibited in vitro in the presence of haemolymph of schistosome-infected snails. This inhibition appeared to be established by a peptidergic factor called schistosomin. The receptor assay was used to identify schistosomin from haemolymph during subsequent purification and characterization steps. The peptide could also be purified from the central nervous systems of non-infected snails, indicating that it is produced by the snail itself and released into the haemolymph as a result of infection. Analysis by plasma-desorption mass spectrometry revealed that purified schistosomin has a molecular mass of 8780 Da.     

The Synthesis and Release of Acetylcholine by Depolarized Hippocampal Slices Is Increased by Increased Choline Available In Vitro Prior to Stimulation

The objective of these experiments was to determine whether preincubating hippocampal slices with choline provides precursor that can be used during a subsequent incubation to support or enhance the synthesis of acetylcholine (ACh). Slices were preincubated for 60 min with 0, 10, 25, or 50 microM choline, washed, resuspended, and then incubated for 10 min in choline-free buffer containing 4.74 (Krebs-Ringer bicarbonate, KRB) or 25 mM KCl. The tissue contents of ACh and choline were determined prior to and after the preincubation, as well as after the incubation; the amounts of ACh and choline released were measured, and ACh synthesis was calculated. Preincubation in the absence of choline increased the tissue content of ACh to 242% of original levels; preincubation with 10 microM choline did not lead to a further increase, but preincubation with 25 or 50 microM choline increased the ACh content to 272% of original levels, significantly greater than that of slices preincubated with either 0 or 10 microM choline. When tissues were subsequently incubated for 10 min with either KRB or 25 mM KCl, ACh release from slices preincubated with 50 microM choline was greater than from slices preincubated with 0, 10, or 25 microM choline. Incubation of slices with KRB did not alter the tissue content of ACh, but when tissues were incubated with 25 mM KCl, the ACh content of slices preincubated with 0 or 10 microM choline decreased significantly, whereas that of slices preincubated with 25 or 50 microM choline did not.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biomphalaria glabrata: influence of calcium, lectins, and plasma factors on in vitro phagocytic behavior of hemocytes of noninfected or Schistosoma mansoni-infected snails.

In vitro phagocytosis of erythrocytes by hemocytes of B. glabrata, intermediate host of S. mansoni, is strongly influenced by calcium, several lectins, and plasma factors. Our results indicate that two different mechanisms of non-self-recognition in B. glabrata may occur: (1) In the presence of calcium, phagocytosis occurs in noninfected and in infected snails without involvement of any other substances, and hemocytes of schistosome resistant as well as those of susceptible snails are able to recognize and phagocytose the target cells. (2) In the absence of calcium, phagocytosis occurs if bridging molecules (heterologous lectins in our assays) were present for which effector and target cells possess binding sites or if target cells were plasma coated prior to the assays. In suspensions in homologous plasma, hemocytes of both snail strains, infected or noninfected, subsequently showed phagocytic activities of about 70-80%. Preincubation of target cells in homologous plasma resulted in similar high phagocytic activities of hemocytes even in the absence of plasma during the standard assay. In these assays, a significantly higher proportion of hemocytes of resistant snails phagocytosed plasma-opsonized erythrocytes, whereas hemocytes of susceptible snails internalized less erythrocytes per cell and needed 60 min to phagocytose at percentages equivalent to that of resistant hemocytes within 10 min. Preincubation of erythrocytes in resistant plasma significantly increased the subsequent phagocytic activity of susceptible hemocytes, whereas preincubation of erythrocytes in susceptible plasma decreased the phagocytosis level of resistant hemocytes.     

Effects of cholesterol evulsion on susceptibility to perfringolysin O of human erythrocytes

Human erythrocytes preincubated with a phosphatidylcholine suspension (preincubated cells) showed decreased susceptibility to perfringolysin O, the decrease being strongly affected by preincubation time and temperature, and the phosphatidyl choline concentration. The binding of the toxin to the preincubated cells also decreased with the preincubation time and reached minimum at 37 degrees C for 6 h. Through this preincubation, about 30% of cholesterol was removed from cells without lysis. The susceptibility of preincubated cells to the toxin seemed to be affected by the amount of cholesterol removed from cells, but not by the cholesterol content of cell membranes. This indicates that most of the cholesterol interactive with the toxin is removable from cell membranes by preincubation with phosphatidylcholine suspension, and that the residual cholesterol is firmly constituted in the membrane structure and cannot interact with the toxin. After cholesterol evulsion by the preincubated plasma method (Murphy, J.R. (1962) J. Lab. Clin. Med. 60, 86-109 and 60, 571-578), cells also exhibited lower susceptibility to the toxin and to saponins, but higher susceptibility to lysophosphatidylcholine.     

beta-arrestin-dependent desensitization of luteinizing hormone/choriogonadotropin receptor is prevented by a synthetic peptide corresponding to the third intracellular loop of the receptor

Desensitization is a ubiquitous response of guanine nucleotide-binding protein-coupled receptors (GPCRs) characterized by the waning of effector activity despite continued presence of agonist. Binding of an arrestin to the activated, often phosphorylated GPCR triggers desensitization. We reported for the luteinizing hormone/choriogonadotropin receptor (LH/CG R) that beta-arrestin tightly bound to porcine ovarian follicular membranes mediates agonist-dependent desensitization of LH/CG R-stimulated adenylyl cyclase (AC) activity (Mukherjee, S., Palczewski, K., Gurevich, V. V., Benovic, J. L., Banga, J. P., and Hunzicker-Dunn, M. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 493-498). We now show that addition of a synthetic peptide corresponding to the entire third intracellular loop (3i) of the LH/CG R completely and specifically reverses desensitization of AC activity, with an ED50 of 10 microM but does not modulate basal, hCG-stimulated, or forskolin-stimulated AC activities. beta-Arrestin binds selectively to the 3i peptide coupled to activated Sepharose. Desensitization of LH/CG R-stimulated AC activity is rescued when the 3i peptide is preincubated with exogenous beta-arrestin. These results show that endogenous beta-arrestin participates in cell-free desensitization of agonist-dependent LH/CG R-stimulated AC activity in follicular membranes by interacting directly with the 3i loop of the receptor, thereby preventing Gs activation.     

Uptake and metabolism of choline in primary isolated neurons and glial cells in culture

The influx and metabolism of choline have been studied in primary cultures of isolated neurons and glial cells from chick embryo dissociated cerebral hemispheres. The results showed a correlation between both influx and metabolism of choline and the exogenous concentrations of choline. When neurons and glial cells were preincubated (10 min) and incubated in Krebs-Ringer phosphate solution with concentrations of choline lower (0.5 μM) or higher (150 μM) than the one present in the growth medium, the metabolism of choline, as a function of time, approached saturation following unusual kinetics. This suggests a non steady state of the endocellular concentrations of free choline. Moreover, when both neurons and glial cells were preincubated (10 min) with 50 μM choline and then incubated (2 min) with various concentrations of choline, only one uptake mechanism was measured, while the preincubation in the absence of choline followed by the incubation of the cells with various concentrations of choline showed the presence of two apparent K_m's with different affinities.The results also indicate the capacity of glial cells to incorporate choline suggesting a storage function for the cells.     

Effect of aminoglycosides on the binding of C14-benzylpenicillin by penicillin-binding proteins in Escherichia coli

It was shown that preincubation of E. coli intact cells with gentamicin and streptomycin induced a marked increase in binding of 14C-benzylpenicillin to its final targets in the membrane i.e. penicillin-binding proteins (PBP). The stimulating effect of the aminoglycosides was also confirmed in experiments with a membrane fraction isolated from the cells preincubated with the aminoglycosides. The PBPs of the cells preincubated with the aminoglycosides were studied with SDS-PAAG electrophoresis. It was revealed that under the action of the aminoglycosides the quantity of the labeled substance (intensity of the bands on the fluorograms) fixed by the low molecular PBPs i.e. D-alanine carboxypeptidases increased. Moreover, the composition of the high molecular less mobile PBPs (transpeptidases) changed. The data are discussed in regard to the peculiarities of the effect of the aminoglycosides on the cells (bactericidal action, membrane tropism). The effect of the aminoglycosides can influence (along with the others) the results of their combined use with beta-lactams.     

Detection of gonadotrophic hormones on isolated thecal interstitial cells of the domestic hen, Gallus domesticus, by an immunohistochemical method

Summary An immunohistochemical method was used to demonstrate the presence of gonadotrophins in isolated ovarian interstitial cells. The cells were obtained by collagenase digestion of large ovarian follicles after removal of the yolk and the granulosa layer. Using a peroxidase-labelled anti-rabbit serum with anti-chicken follicle stimulating hormone (FSH) serum raised in rabbits, a strong positive reaction was obtained. Anti-human FSH serum also produced a positive result but the reaction was weaker. There was no apparent difference in the staining reaction of cells which had been preincubated with ovine FSH serum. Treatment with anti-ovine luteinizing hormone (LH) resulted in a faintly positive reaction.The viability of the cells was tested by the Trypan Blue method and they were identified as steroid-producing cells by the histochemical demonstration of their 3-hydroxysteroid dehydrogenase activity.     

Prior serum- and AICAR-induced AMPK activation in primary human myocytes does not lead to subsequent increase in insulin-stimulated glucose uptake

Exposing isolated rat skeletal muscle to 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside [AICAR, a pharmacological activator of AMP-activated protein kinase (AMPK)] plus serum leads to a subsequent increase in insulin-stimulated glucose transport (Fisher JS, Gao J, Han DH, Holloszy JO, and Nolte LA. Am J Physiol Endocrinol Metab 282: E18-E23, 2002). Our goal was to determine whether preincubation of primary human skeletal muscle cells with human serum and AICAR (Serum+AICAR) would also induce a subsequent elevation in insulin-stimulated glucose uptake. Cells were preincubated for 1 h under 4 conditions: 1) without AICAR or serum (Control), 2) with serum, 3) with AICAR, or 4) with Serum+AICAR. Some cells were then collected for immunoblot analysis to assess phosphorylation of AMPK (pAMPK) and its substrate acetyl-CoA carboxylase (ACC). Other cells were incubated for an additional 4 h without AICAR or serum and then used to measure basal or insulin-stimulated 2-deoxyglucose (2-DG) uptake. Level of pAMPK was increased (P < 0.01) for myotubes exposed to Serum+AICAR vs. all other groups. Phosphorylated ACC (pACC) levels were higher for both Serum+AICAR (P < 0.05) and AICAR (P < 0.05) vs. Control and Serum groups. Basal (P < 0.05) and 1.2 nM insulin-stimulated (P < 0.005) 2-DG uptake was higher for Serum vs. all other preincubation conditions at equal insulin concentration. Regardless of insulin concentration (0, 1.2, or 18 nM), 2-DG was unaltered in cells preincubated with Serum+AICAR vs. Control cells. In contrast to results with isolated rat skeletal muscle, increasing the pAMPK and pACC in human myocytes via preincubation with Serum+AICAR was insufficient to lead to a subsequent enhancement in insulin-stimulated glucose uptake.     

Conserved form and function of the germinal epithelium through 500 million years of vertebrate evolution

The germinal epithelium, i.e., the site of germ cell production in males and females, has maintained a constant form and function throughout 500 million years of vertebrate evolution. The distinguishing characteristic of germinal epithelia among all vertebrates, males, and females, is the presence of germ cells among somatic epithelial cells. The somatic epithelial cells, Sertoli cells in males or follicle (granulosa) cells in females, encompass and isolate germ cells. Morphology of all vertebrate germinal epithelia conforms to the standard definition of an epithelium: epithelial cells are interconnected, border a body surface or lumen, are avascular and are supported by a basement membrane. Variation in morphology of gonads, which develop from the germinal epithelium, is correlated with the evolution of reproductive modes. In hagfishes, lampreys, and elasmobranchs, the germinal epithelia of males produce spermatocysts. A major rearrangement of testis morphology diagnoses osteichthyans: the spermatocysts are arranged in tubules or lobules. In protogynous (female to male) sex reversal in teleost fishes, female germinal epithelial cells (prefollicle cells) and oogonia transform into the first male somatic cells (Sertoli cells) and spermatogonia in the developing testis lobules. This common origin of cell types from the germinal epithelium in fishes with protogynous sex reversal supports the homology of Sertoli cells and follicle cells. Spermatogenesis in amphibians develops within spermatocysts in testis lobules. In amniotes vertebrates, the testis is composed of seminiferous tubules wherein spermatogenesis occurs radially. Emerging research indicates that some mammals do not have lifetime determinate fecundity. The fact emerged that germinal epithelia occur in the gonads of all vertebrates examined herein of both sexes and has the same form and function across all vertebrate taxa. Continued study of the form and function of the germinal epithelium in vertebrates will increasingly clarify our understanding of vertebrate reproduction. J. Morphol. 277:1014–1044, 2016. © 2016 Wiley Periodicals, Inc.     

Induction of sexual co-flocculation of heterothallic fission yeast (Schizosaccharomyces pombe) cells by mating pheromones

Heterothallic fission yeast (Schizosaccharomyces pombe) cells preincubated with sex pheromone, P- or M-factor of the obverse mating-type cells, in mannose synthetic medium (MSM) results in remarkably increased sexual co-flocculation with obverse mating-type cells almost without time lag, i.e., within 10 min. By contrast, comparable flocculation requires over 1 h if untreated control cells are mixed with obverse mating-type cells. The agglutinin of P cells is more inducible than that of M cells. These pheromonal inductions of sexual co-flocculation are inhibited by the addition of cycloheximide or tunicamycin during preincubation but not by chloramphenicol or hydroxyurea. These results demonstrate that, in addition to (a) the repression of cell division (G1 arrest) and (b) the activation of cell wall autolytic processes (mating-specific elongation of cells: formation of their conjugation tubes), mating pheromones of fission yeast have another important role; (c) to induce sexual co-flocculation (agglutinability). Using our experimental system of preincubation with sexual pheromones, we show that M-agglutinin is heat-stable and its induction is inhibited by tunicamycin, but that P-agglutinin is heat-labile and its induction is only partially inhibited by tunicamycin.     

Gynandromorphs of Drosophila suggest one common primordium for the somatic cells of the female and male gonads in the region of abdominal segments 4 and 5.

In mosaic gonads of gynandromorphs of Drosophila, the amount of female and the amount of male somatic tissues add up to roughly one unit. This suggests that the somatic component of the gonads in males and females derives from a single common primordium, i.e. testes and ovaries appear to be homologous. Fate-mapping places this primordium ventrally of the sternites into the mesodermal region of the fourth and fifth abdominal segment. This location is corroborated by the observation that defects in and around abdominal segment 4 and absence of the gonads are strongly correlated in animals damaged by the mutation osk301. Gonads were mosaic with a frequency of 10.5% which indicates that the gonadal primordium originates from about 10 progenitor cells, and together with other evidence, suggests that these progenitor cells are located within a single segment (or parasegment).     

Effect of Arginine-deficient Media on the Herpes-Type Virus Associated with Cultured Burkitt Tumor Cells

After incubation at 37 C for 2 or more days, Basal Medium Eagle (BME) supplemented with 25% fetal calf serum (FCS) was not able to support adequate growth of EB3 and other lines of Burkitt tumor cells. The medium did, however, increase, by a factor of about 10, the number of cells synthesizing herpes-type virus with which the cultures were persistently infected. Not every lot of FCS produced these effects, nor were these effects seen when BME and FCS were incubated separately for 7 days before the medium was completed. At 37 C, appropriate lots of FCS interacted with several of the amino acids present in BME; this interaction resulted in an inhibition of cellular growth, whereas interaction with arginine yielded the virus-enhancing effect. Arginine-free BME, supplemented with 25% FCS and used without prior incubation, prevented cellular replication and promoted viral infection to a similar extent as did preincubated complete medium. Replenishment of arginine reduced, but did not regularly abolish, the virus-enhancing activity of preincubated media. RPMI-1629 medium was less affected by preincubation with FCS because it contained twice the amount of arginine that BME contained. The FCS factors which act upon arginine and other amino acids are not dialyzable and are partially resistant to heating at 56 or 60 C for 30 to 60 min. Calf and horse sera appear to be devoid of these activities. The nature of these interactions, as well as the mechanism by which arginine deficiency enhanced the viral infection, remains to be ascertained.     

A 31P NMR study on uptake and metabolism of hexose monophosphates in rat diaphragm muscle

Phosphorus nuclear magnetic resonance spectroscopy was used to study uptake and metabolic conversion of glycolytic intermediates in rat diaphragm muscles. The resonances of several phosphorus-containing metabolites were identified in the intact tissues and in their ethanolic extracts. Experiments on muscles preincubated with glucose 6-phosphate or glucose 1-phosphate indicated that: 1) both substrates penetrate into the tissue and actively participate in glycolytic reactions; 2) glucose 1-phosphate is completely converted into other metabolites, including glucose 6-phosphate and its products; 3) preincubation with either hexose monophosphate in the presence or in the absence of insulin produced the same set of phosphorylated metabolites. Addition of insulin to preincubation media induced a conspicuous spread of chemical shifts in the band arising from phosphorylated sugars in tissues incubated with glucose 1-phosphate but not in those treated with glucose 6-phosphate. The different responses to insulin exhibited by the 31P n.m.r. spectral profiles of tissues incubated with the two substrates substantiate the hypothesis that glucose 6-phosphate and its products may undergo their metabolic conversions in the tissue along distinct intracellular enzymatic pathways, on which insulin would exert different regulatory effects. This study indicates that 31P n.m.r. may provide a useful approach to the elucidation of metabolic processes involving sugar phosphates in intact tissues.     

Generation of suppressor cells by concanavalin A: a new perspective

Significantly lower mitogenic responses of fresh cells co-cultured with Con A-stimulated cells were found when compared with the responses of fresh cells co-cultured with preincubated control cells. We do not agree with the interpretation that this effect represents the generation of suppressor cells by Con A, since the responses of fresh cells cultured alone were also significantly less than when co-cultured with control cells and the same as when co-cultured with the Con A-stimulated cells. Treatment with mitomycin C was sufficient to prevent the preincubated cells from contributing to the mitogenic response of the fresh cells. The increased responses of fresh cells when co-cultured with preincubated cells seems analagous to the increased mitogenic responses of cells aged in vitro by preincubation without mitogen. This effect seems to be transferable to fresh cells in the absence of cell division. Although preincubation in the presence of Con A abrogates this effect, we do not interpret this as the generation of suppressor cells.