Synthesis of two storage proteins during larval development of the tobacco hornworm, Manduca sexta

Studies of synthesis and accumulation of the two storage proteins arylphorin and female-specific protein (FSP) during the final two larval instars of the tobacco hornworm showed both stage and temporal specificity. Arylphorin was present in both stages, but its synthesis ceased during the molt, during starvation, and at the wandering stage, and then resumed about 24 hr after the onset of feeding. During the larval molt about 25% of injected iodinated arylphorin was incorporated into the newly forming fifth instar cuticle. The cessation of arylphorin synthesis was mimicked by exposure of the fat body to 1 microgram/ml 20-hydroxyecdysone (20HE) in complete Grace's medium or to dilutions of Grace's medium greater than 50%. Lower concentrations of 20HE were ineffective, indicating that the cessation of synthesis in vivo was likely due to a combination of lack of excess nutrients and the hormonal milieu. The female-specific protein was not synthesized until the final larval instar, appearing first in females on Day 2 and later in males at the time of wandering, with synthesis continuing throughout the prepupal period. In vitro studies showed that this protein was synthesized as a 620-kDa protein, and then during secretion a 730-kDa immunoreactive form also appeared. Synthesis of FSP was inhibited by exposure of Day 2 fat body to 1 microgram/ml 20HE for 24 hr. Ligation followed by 20HE infusion showed that the disappearance of FSP from the hemolymph during the prepupal period was controlled by the rising ecdysteroid titer.     

Kinetics of CTL induction by concanavalin A.

Induction of maximal CTL activity was achieved within 12 hr of exposure to Con A in vitro in various mouse lymphoid cell populations. These included spleen cells from normal unsensitized mice, spleen cells from mice previously immunized with alloantigen, and mouse spleen cells exposed to alloantigen in long-term mixed leukocyte culture (LTMLC). Although induction of maximal incorporation of tritiated thymidine was accomplished within this same period in the cells obtained from LTMLC, a much longer period of Con A exposure (greater than 24 hr) was required for freshly prepared spleen cells from normal or previously immunized mice. These findings indicate that the increased tritiated thymidine uptake induced in freshly prepared spleen cells on continued exposure to Con A beyond 12 hr is not associated with the development of cytolytic activity, and that it probably represents stimulation of subpopulations no longer present in the LTMLC population where positive selection for cells responsive to cellular alloantigens has taken place.     

Distribution of endotoxin and its effect on serum concentrations of C-reactive protein and cortisol in the plaice (Pleuronectes platessa L.)

1. Bacterial endotoxins cause a significant increase in the serum concentration of both C-reactive protein (CRP) and cortisol in the plaice.2. The time course of the CRP elevation varies with the bacterial source, method of isolation of the endotoxin and the route of administration.3. Injection of the prostaglandin synthetase inhibitor, indomethacin, 2 hr before endotoxin, abolishes the increases in both CRP and cortisol, whereas it has no effect when given 6 hr after.4. Using [⁵¹Cr] labeled endotoxin, 40% of the total injected dose was localized in the kidney and 36% in the spleen, 24 hr after i.v. injection. Uptake was five times greater per g of spleen than of kidney.     

The interaction of the steroidal antagonist faslodex and methotrexate.

Faslodex (FAS, ICI 182, 780), a novel steroidal estrogen antagonist decreased high-dose methotrexate (MTX) cytotoxicity in MCF-7 breast cancer cells. When FAS is given at least 24 hr prior to MTX, the resultant interaction is antagonistic. However, when breast cancer cells are exposed to FAS 24 hr after MTX, the interaction between FAS and MTX is not antagonistic. The proliferation of cells exposed to 0.1 microM FAS and 10 microM MTX alone or in combination with FAS 24 hr prior to MTX was in the following order: FAS>FAS 24 hr prior to MTX>MTX. MTX administration 24 hr prior to FAS had the following inhibitory effects on the growth of cells: MTX 24 hr prior to FAS >MTX>FAS 24 hr prior to MTX>FAS>control (no drug exposure). To determine if the antagonistic interaction between FAS and MTX was a function of sequence and time, cells were exposed to FAS 24 hr and 36 hr prior to MTX exposure. The percentages of control rates were 42.70 +/- 4.60% and 57.89 +/- 0.55%, respectively, from a 24 hr and 36 hr exposure of FAS prior to MTX. The growth rates after 24 and 36 hr exposures to MTX alone were 30.30 +/- 0.61% and 33.11 +/- 2.57% of control rates, respectively. These studies suggest that: a) the interactions between FAS and MTX are sequence-dependent; b) FAS antagonizes the effect of MTX when FAS administration precedes MTX, and c) FAS antagonism to MTX is a function of time.     

Tolbutamide alters glucose transport and metabolism in the embryonic mouse heart

BACKGROUND: Tolbutamide is a sulfonylurea oral hypoglycemic agent widely used for the treatment of non insulin-dependent diabetes mellitus. Tolbutamide produces dysmorphogenesis in rodent embryos and becomes concentrated in the embryonic heart after maternal oral dosing. Tolbutamide increases glucose metabolism in extra-pancreatic adult tissues, but this has not previously been examined in embryonic heart. METHODS: CD-1 mouse embryos were exposed on GD 9.5 to tolbutamide (0, 100, 250, or 500 microg/ml) for 6, 12, or 24 hr in whole-embryo culture. Isolated hearts were evaluated for (3)H-2DG uptake and conversion of (14)C-glucose to (14)C-lactate. Glut-1, HKI, and GRP78 protein levels were determined by Western analysis, and Glut-1 mRNA was measured by RT-PCR. RESULTS: Cardiac (3)H-2DG uptake increased after exposure to 500 microg/ml tolbutamide for 6 hr, and 100, 250, or 500 microg/ml tolbutamide for 24 hr, compared to controls. Glycolysis increased after exposure to 500 microg/ml tolbutamide for 6 or 24 hr compared to controls. Glut-1 protein levels increased in hearts exposed to 500 microg/ml tolbutamide for 12 or 24 hr, and Glut-1 mRNA increased in hearts exposed to 500 microg/ml tolbutamide for 24 hr compared to controls. HKI protein levels increased in hearts exposed to 500 microg/ml tolbutamide for 6 hr, but not 12 or 24 hr. There was no effect on GRP78 protein levels in hearts exposed to tolbutamide for 6, 12, or 24 hr. CONCLUSIONS: Tolbutamide stimulates glucose uptake and metabolism in the embryonic heart, as occurs in adult extra-pancreatic tissues. Glut-1 and HKI, but not GRP78, are likely involved in tolbutamide-induced cardiac dysmorphogenesis.     

Effect of cobalt on heme biosynthesis in rat liver and spleen.

1. Succinate-cytochrome c reductase activity in rat liver decreased to about 60% of the control value after a single injection of cobalt or in a steady state of intoxication, but the activity in the spleen was unaltered. 2. Incorporation of radioactive glycine and 5-aminolevulinate into heme of the liver was markedly inhibited by cobalt treatment. 3. 5-Aminolevulinate synthase [EC 2.3.1.37] activity in the liver decreased to 40% of the control value 4 hr after cobalt injection, and completely recovered 20 hr later. Phenylhydrazine-induced 5-aminolevulinate synthase activity in the spleen was not decreased by cobalt injection. 4. Porphobilinogen synthase [EC 4.2.1.24] activity in the liver decreased and reached its minimum value (42% of the control) 12 hr after cobalt injection. On the other hand, the activity in the spleen showed a marked increase 24 hr after coblat injection. 5. Ferrochelatase [EC 4.99.1.1] activity in the liver was essentially unaltered by cobalt treatment, while the activity in the spleen was elevated dramatically after 24 hr. 6. Concentrations of cobalt after a single injection were about 0.3 mM and 0.03 mM in the liver and spleen, respectively. 7. Inhibitions of 5-aminolevulinate synthase and porphobilinogen synthase activities by cobalt in vitro were not as marked as expected from in vivo experiments.     

Dietary regulation of adenosine deaminase activity in stomach, small intestine and spleen of mice

Activity of adenosine deaminase (ADA) and its regulation by dietary restriction were studied in the stomach, small intestine and spleen of mice. ADA activity (U/mg protein) was highest in the stomach, followed by small intestine and spleen of mice on normal diet. The activity decreased significantly in the stomach (41%) and small intestine (45%) of 24 hr fasted mice, when compared to mice fed ad-libitum. However, ADA activity in spleen did not show any change by dietary intervention. Refeeding of fasted mice for 24 hr restored the activity of ADA in tissues. In addition, dietary restriction (alternate days of feeding for three months) had a cumulative effect, whereby ADA activity decreased significantly in the stomach (53% on the day of feeding and 60% on the day of fasting) and small intestine (50% and 54% on the day of feeding and fasting, respectively) without any change in activity in spleen. These findings indicate that dietary restriction reduces ADA activity in a tissue-specific manner. Long-term dietary restriction leads to a cumulative adaptation in lowering the ADA activity of GIT, but not in spleen.     

B-naphthoflavone induction and its effect on hepatic phospholipid metabolism in rainbow trout (Salmo gairdneri)

1. B-naphthoflavone (BNF) induction of microsomal cytochrome P-488 and its effect on liver phospholipid metabolism were examined in rainbow trout (Salmo gairdneri) 24 and 96 hr after intraperitoneal injection. 2. Cytochrome P-448 content increased at 96 hr to approximately double the cytochrome content of control fish. 3. Computer-analyzed laser densitometry scans of LDS-polyacrylamide gels of 96 hr BNF-treated liver microsomes showed a 90% increase in cytochrome P-448 levels. 4. A 34% increase in microsomal phospholipid (mumol/mg protein) was observed 24 hr after BNF injection, with a marked increase in choline, ethanolamine and inositol phospholipids. 5. Following 96 hr of exposure to BNF some differences in enzyme activity were noted; choline kinase and cytidylyltransferase activities were reduced, while a marked increase was observed in choline phosphotransferase activity. In light of current information on induction of liver microsomal phospholipid metabolism in mammals, the results of the this study suggest that trout do not respond like mammals to inducers of monooxygenase activity.     

The effect of dexamethasone on the cytotoxic and enzymatic response of cultured endothelial cells to radiation

Experiments were conducted to determine (1) whether glucocorticoids directly protected endothelial cells (EC) from radiation and (2) if angiotensin converting enzyme (ACE) activity, known to be increased by glucocorticoid, played a role in the EC response to radiation. Confluent monolayers of EC cultured from bovine aorta EC were treated with dexamethasone (10(-6) M); after irradiation (5.0 Gy, 60Co gamma), ACE and lactate dehydrogenase (LDH) activities, DNA and protein contents, and nuclei number were measured. Twenty-four hours after 5 Gy, there was increased cell loss (-40%, P less than 0.001), greater LDH release (greater than 100%, P less than 0.001), more LDH activity per cell (+40%, P less than 0.001), and unchanged ACE activity compared to sham-irradiated control EC. However, 48 hr after 5 Gy, ACE activity per cell was decreased (-24%, P less than 0.005). A 48-hr exposure to dexamethasone alone was accompanied by a slight cell loss (-10%, P less than 0.001) and increased cellular ACE activity (+40-140%, P less than 0.001), but a 24-hr dexamethasone exposure was not cytotoxic and did not change ACE activity. Dexamethasone exposure for 48 hr before and after irradiation did not attenuate cell loss or LDH release. However, combined dexamethasone treatment and radiation increased cellular ACE activity at a time when neither agent alone had an effect (24-hr dexamethasone exposure before 5 Gy and assayed 24 hr after 5 Gy). This interaction between radiation and dexamethasone treatment suggests that the glucocorticoid modifies the cell's response to injury. Although this interaction does not ameliorate radiation cytotoxicity, maintenance of ACE levels in injured vessels by hormones may have physiological significance in the hemodynamics of irradiated tissues.     

Effect of subacute exposure to methyl isocyanate on testicular histomorphology in mice

Mice exposed to methyl isocyanate (MIC; 134 mg.m-3 for 30 min = 4020 mg.min-1.m-3) showed a marked loss of body weight after 24 hr and the mean body weight of the exposed group was significantly less than the control, even 15 days after the exposure. No significant change was observed on relative testicular weight. Spermatozoa in the seminiferous tubules disappeared 3 days post exposure. Primary and secondary spermatocytes were hypertrophied. Normalization occurred after 15 days.     

Increased serum enzyme activity in microwave-exposed rats

Heat stable serum enzymes were studied in rats exposed to microwaves (2.45 GHz, 120 Hz amplitude modulated) 24 hr after a single 4-hr exposure or immediately after 3 and 10 exposures to 0.1 to 55 mW/cm2. In addition, stable colonic temperature at 41.5 degrees C for 30 min was maintained by microwave exposure in a group of five rats under barbiturate anesthesia. Alkaline phosphatase and lactic dehydrogenase did not increase as a result of microwave exposure. Increased serum glutamic pyruvic transaminase (GPT) and glutamic oxaloacetic transaminase (GOT) were noted in the 41.5 degrees C group 24 hr after exposure. A threshold body temperature for acute cellular injury after microwave exposure was demonstrated. The acute cellular injury could be in the liver. These mild elevations in the serum enzyme levels (mean +/- SE, GOT = 167 +/- 40 U/liter: GPT = 74 +/- 26 U/liter) indicated that the injuries were not accompanied by any significant sequelae in the rat. From this threshold and colonic temperature (41.5 degrees C for 30 min) in barbiturate-anesthetized, microwave-exposed rats, we derived a tentative threshold for the whole-body average absorption rate at 14 W/kg (70 mW/cm2 at 2.45 GHz for adult rats) for 4 hr. This tentative threshold is subject to changes by duration of exposure and by compounding variables influencing maintenance of body temperature.     

Effects of storage and incubation conditions on human granulocyte phagocytic, bactericidal, and chemotactic functions

This study was undertaken to determine the effects of different incubation conditions on human granulocyte (PMN) bactericidal, phagocytic, and chemotactic functions. Specifically, (1) how long may a patient's blood be held before assay and maintain original PMN function, and (2) how long may isolated PMNs be incubated for the purpose of exposure to various agents and still maintain original function? PMNs isolated following storage of whole heparinized blood at 4 °C for 24 and 48 hr phagocytized as well as fresh cells and their bactericidal activity was 96 and 85% of control values after 24 and 48 hr, respectively. Chemotaxis decreased to 62% of control after 24 hr. The bactericidal capacity of isolated PMNs stored at 4 °C for 24, 48, and 72 hr decreased to 85, 81, and 78% of controls, respectively. Phagocytosis after 24 hr storage was equal to controls. Chemotaxis was decreased to 59 and 34% of controls after 24 and 48 hr, respectively. Isolated PMNs incubated at 37 °C demonstrated impairment in phagocytic capacity after only 4 hr.     

In vivo adaptive control of beta-receptors and adenylate cyclase during short-term heat exposure in rat parotid glands.

1. Adaptation of beta-adrenergic receptors (beta-AR) and adenylate cyclase (AC) in rat parotid glands during short-term heat exposure (33 degrees C) were studied. 2. Heat exposure reduced AC activity in response to isoproterenol (IPR). 3. The number of beta-AR on the cell surface significantly increased after 24 hr but returned to control level after 48 hr. 4. IPR-induced [3H]GDP release was significantly reduced throughout exposure. 5. The data suggest that the major factor which results in the desensitization of AC during short-term heat exposure is a blunted coupling between beta-AR and GTP binding protein(s).     

Suppression of the in vitro secondary antibody response of rabbit lymphoid cells by Concanavalin A.

The effect of various concentrations of concanavalin A (Con A) on the in vitro secondary antibody response of rabbit lymph node and spleen cells to sheep red blood cells (SRBC) was studied. Complete suppression of the IgM plaque-forming cell (PFC) response of both lymph node and spleen cultures was observed when 10 mug/ml of Con A was added at the time of initiation of the cultures whereas only partial suppression was observed when 1 mug/ml of Con A was added. Moreover, marked suppression of the immune responses of both spleen and lymph node cultures was observed when 10 mug/ml of Con A was added at 24 hr after antigenic challenge and to a lesser extent when added at 48 hr. Suppression of the IgM PFC response was also detected when spleen cultures were exposed to 10 mug/ml of Con A for as little as 2 hr after antigenic challenge. However, substantial increases in DNA synthesis were observed only in those cultures which were in contact with Con A for at least 24 hr. Finally evidence is presented that the Con A-induced suppression is mediated by a soluble substance(s).     

Studies on the regulation of lymphocyte production in the murine thymus and some effects of a crude thymus extract.

Cell proliferation in the murine thymus was studied in vivo under normal conditions and from 0 to 24 hr after a single injection of a water-soluble extract from mouse thymus, mouse spleen, and mouse skin. The thymus extract reduced during the first 24 hr the mitiotic activity 40%; the spleen extract had a weaker inhibitory effect. The skin extract had no such effect. The thymus extract and spleen extract inhibited the flux of cells into the S phase 0-8 hr after the injection of the extract. Initial labelling index was also reduced in this period. Eight hours after injection of the thymus or spleen extracts the inhibited cells initiated DNA synthesis. The rate of progression of blast cells through the cell cycle was normal 24 hr after the injection of the extracts. It was deduced from the analysis that the thymus extract inhibits processes triggering G0/G1 cells into DNA synthesis, the inhibition of G2 efflux being of minor importance. Finally a model for the regulation of proliferating thymic blast cells and the emigration of small lymphocytes from the thymus is proposed.     

Modulation of mitogen-induced spleen cell proliferation and the antibody-forming cell response by beta-endorphin in vivo

Experiments were conducted which compared the in vivo effects of beta-endorphin (BEP), gamma-endorphin (gamma EP), methionine-enkephalin (Met-ENK), and acetylated BEP(1-27) on the in vitro proliferative response of rat spleen cells to concanavalin A (ConA). In addition, the influence of BEP administration on the primary and secondary antibody-forming cell (AFC) response to the soluble antigen keyhole-limpet hemocyanin (KLH) was examined. Intravenous administration of BEP enhanced the spleen cell proliferative response to ConA assessed 3 hr after a single bolus infusion. Conversely, infusion with AcBEP(1-27) suppressed the proliferative response, whereas no effects of intravenous gamma EP or Met-ENK treatment were observed. The enhancing effect of BEP administration was not detectable 24 hr after a single infusion, but could be maintained over a 44 hr period by multiple infusions. The primary AFC response to KLH was suppressed by a dose of 1 nmole BEP only. On the other hand, the secondary IgG AFC response to KLH was enhanced by 10 pmoles BEP, while the IgM and IgA AFC responses remained unaltered by BEP treatment. The anamnestic in vitro proliferative response of spleen cells cultured with KLH was not altered if BEP was administered at the time of secondary KLH immunization. These results extend previous observations of BEP-induced modulation of in vitro immune function by demonstrating that opioid and nonopioid forms of BEP administered in vivo alter the capacity of spleen cells to proliferate and develop antibody responses to antigen.     

Acute toxicity and bioaccumulation of endosulfan in rotifer (Brachionus calyciflorus).

1. The acute toxicity of endosulfan was determined for the freshwater rotifer Brachionus calyciflorus. 2. The mean 24 hr LC50 value for endosulfan was 5.15 ppm with a coefficient of variation of 14.7%. 3. Rotifers were exposed at two sublethal concentrations (1.5-2.0 ppm) of endosulfan for bioaccumulation experiments, for an exposure time of 24, 48, 72 and 96 hr. The rotifers were fed with Nannochloris oculata (5 x 10(5) cell/ml). 4. The highest accumulation of endosulfan was found 24 hr after the start of the exposure to 1.5 ppm of the toxicant. A steady-state concentration in rotifer was reached between 24-48 hr, followed by a gradual decrease until 96 hr.     

Regulation of the immune system by synthetic polynucleotides. VII. Suppression induced by pretreatment with poly A:U.

Pretreatment of mouse spleen cells with polyadenylic-polyuridylic acid complexes (poly A:U) either in vivo or in vitro 24 hr prior to addition of antigen, resulted in a substantial time dependent decrease in anti-SRBC PFC. Enhancement was observed 6 hr after poly A:U, while inhibition did not become evident until 24 hr after pretreatment. Inhibition of the PFC response appeared to result from poly A:U activation of a nylon wool adherent, T suppressor cell, capable of diminishing the response of normal spleen cells exposed to antigen on co-culture.     

Role of protein synthesis in the activation of cytotoxic mouse macrophages by lymphokines

The role of protein synthesis during the activation of macrophages (M phi) by lymphokines (LK) was studied. Peritoneal murine macrophages elicited by proteose-peptone (pM phi) were activated with LK (supernatants from normal mouse spleen cells pulsed with concanavalin A) and tested for cytotoxicity in an 18 hr assay against 111In-labeled L5178Y lymphoma target cells. Reversible (cycloheximide and puromycin) or poorly reversible (emetine and pactamycin) inhibitors of protein synthesis were added during activation, and their effects on pM phi-mediated cytotoxicity and pM phi protein synthesis were measured. Minimal concentrations of inhibitors, reducing the rate of protein synthesis by more than 90% without toxic effects on macrophages, were chosen. Exposure of pM phi to LK for 2 to 18 hr in the presence of reversible inhibitors of protein synthesis did not affect the induction of cytolytic activity, indicating that protein synthesis was not required during the activation period. In contrast, activation of macrophages for 2 hr in the presence of poorly reversible inhibitors of protein synthesis resulted in a considerable reduction of cytolytic activity. The impairment of cytotoxic activity was also evident when pM phi were treated with such drugs during the first 2 hr of an 18 hr exposure to LK or when LK-activated macrophages were treated for 2 hr with the drugs before the addition of the targets. These results demonstrate that active protein synthesis is not required during the exposure of pM phi to LK, but that new proteins have to be synthesized to allow the expression of the cytotoxic activity in LK-activated pM phi.