Stimulatory Substance Research Articles

Factors influencing zoospore production by Phytophthora cinnamomi in axenic cultui

Factors and procedures found to increase the quantity and consistency of axenic zoospore production in a selected isolate of Phytophthora cinnamomi were (i) the use of single-zoospore cultures of uniform size that were between 48 and 72 h old; (ii) thorough washing of mycelial mats at the time of sporangium induction to remove nutrients; (iii) agitation of the sporulation medium (mineral salt solution) 24 h after the initial induction; (iv) standardization of the volume of the sporulation medium; (v) adequate removal of the sporulation medium and replacement with distilled water before triggering zoospore release; and (vi) placement of colonies that had been induced to sporulate under light. The addition of a purified sporangium stimulatory substance to mycelial mats which had been induced to sporulate enabled the fungus to sporulate under conditions which normally suppressed sporulation in vitro. In the presence of this stimulatory substance, the fungus sporulated prolifically in darkness and with limited quantities of added nutrients. Other isolates of P. cinnamomi responded in a similar manner to many of these factors and procedures.

The intestinal phase hormone.

The existence of a stimulatory intestinal phase of gastric acid secretion has been suspected, but largely ignored, for many years. Recently, however, it has become clear that the intestinal phase plays an important role in acid production during digestion. The intestinal phase is of additional interest in relation to the profound gastric acid hypersecretion associated with portacaval shunt (PCS). Substantial evidence indicates that PCS-related gastric hypersecretion is due to unmasking of the intestinal phase by hepatic bypass of a humoral stimulant in portal blood that is normally degraded to a considerable extent by the liver. Studies in our laboratory during the past 12 years have provided strong physiologic evidence for humoral mediation of both the intestinal phase of gastric secretion and of PCS-related hypersecretion by a hormone that arises in the small intestine, particularly in the jejunum. Furthermore, our studies have demonstrated that this intestinal phase hormone (IPH) exists in humans as well as in dogs, rats, and pigs. Additionally, recent work by a number of investigators, as well as by our group, has provided convincing evidence that IPH is different from any of the known gastric stimulatory hormones. With these physiologic observations as a background, we have used a classical method for extracting acidic peptides to prepare a hog intestinal mucosa extract (HIME) that has all of the known physiologic properties of an IPH. Specifically, HIME contains a potent stimulant of gastric acid secretion that acts according to a linear dose-response relationship; that is not gastrin in any of its immunoassayable forms; that significantly augments the maximal acid secretory responses to pentagastrin, gastrin, CCK, and histamine; and that is substantially degraded by the liver, in contrast to gastrin and CCK. Efforts at isolating the gastric stimulatory substance in HIME suggest that it is a peptide of low molecular weight. Work directed at isolating IPH in pure form and identifying it is in progress.

Stimulators and Inhibitors of Lymphocyte DNA Synthesis in Supernatants from Human Lymphoid Cell Lines

Some T and B lymphoid cell lines (LCL) were found to secrete into their supernatants a substance able to stimulate lymphocyte proliferation. This substance produced an increase in [3H]thymidine uptake by mononuclear cells when added to unstimulated cultures (mitogenic effect) or when added to cultures stimulated with phytohemagglutinin (PHA) or pokeweed mitogen (PWM) (potentiating effect). When complete supernatants were used, the potentiating effect was sometimes masked by an inhibitor of DNA synthesis. Fractionation on Sephadex G-100 separated these two activities. The stimulatory substance eluted at a m.w. range of 15,000 to 30,000, and the inhibitor eluted with the albumin peak. B cells with or without monocytes were the most sensitive to the mitogenic effect, whereas T cells were unaffected. Responses to PHA and PWM were potentiated when T cells were present, but the maximum effect was observed when the proportion of T cells was less than 50%. The stimulatory material may be similar to lymphocyte mitogenic factor and may function as a T cell-replacing factor in B cell stimulation.

Isolation of a stimulatory factor for nuclear DNA replication

Aqueous extracts of isolated nuclei and intact plasmodia of Physarum contain a heat-stable stimulator of nuclear DNA replication. The stimulatory factor is present throughout the mitotic cycle, and its activity is unaffected by prior exposure of plasmodia to cycloheximide. The stimulatory substance has been partially purified by heat treatment, precipitation with ethanol, chromatography on DEAE cellulose, and gel filtration. The purified material contains both carbohydrate and protein, and exhibits a molecular weight of about 30 000. The active substance increases the rate and overall extent of DNA replication in S-phase nuclei, but does not trigger the initiation of DNA synthesis in nuclei isolated from G 2-phase plasmodia. The stimulatory material contains little or no deoxyribonuclease or DNA polymerase activity, and it does not affect DNA polymerase activity assayed using a purified DNA template.

Proteolytic activation of rat liver adenylate cyclase by a contaminant of crude collagenase from Clostridium histolyticum.

Treatment of rat liver plasma membranes with various commercial preparations of crude collagenase from Clostridium histolyticum at concentrations as low as 1 mug/ml, resulted in activation of the adenylate cyclase system. Maximal activation occurred at 50 to 100 mug/ml of collagenase, and promoted a 2- to 3-fold increase in the basal activity as well as in the activities stimulated by catecholamines, glucagon, fluoride, or GTP. This was due to an increase in the maximal velocity of the cyclizing reaction without any increase in the affinity of the enzyme for its substrate. Treatment of plasma membranes with crude collagenase did not induce gross structural modifications as judged by electron microscopic examination. 5'-Nucleotidase activity was slightly inhibited and ATPase activity remained unaffected. The stimulatory substance was nondialyzable, thermolabile, and inhibited by both EDTA and -SH reagents, thus appearing to be a protein. The following observations suggest the effects observed were due to other protease(s) present in crude collagenase: (a) only crude collagenase was active on liver adenylate cyclase: treatment with purified collagenase from C. histolyticum or from Achromobacter iophagus gave no stimulation; (b) the stimulatory activity was irreversible since washing of the membranes after treatment was without effect; (c) crude collagenase contained no lecithinase or sphingomyelinase activity under our conditions of adenylate cyclase assay; (d) after chromatography on Sephadex G-100, the activator appeared as a peak in the 30,000-dalton region and was clearly separated from the collagenase and clostripain peaks, but coincident with elastolytic and caseinolytic activities; (e) the effect of crude collagenase could be prevented by addition of elastin in vitro and was mimicked by purified elastase from hog pancreas. It remains to be seen whether the effects observed result from an increase in the catalytic constant of adenylate cyclase, or an unmasking of new catalytic sites.

Mitosis in the intact and regenerating planarian Dugesia mediterranea n.sp. II. Mitotic studies during regeneration, and a possible mechanism of blastema formation

AbstractIn planarian regeneration mitosis occurs in both anterior and posterior blastemas. The mitotic increase is visible as early as one hour after amputation, reaches a rapid first maximum at 5–12 hours, a relative minimum at 24–30 hours, and a higher and longer second maximum at two to four days depending on body size, level of amputation, and on whether blastema is anterior or posterior. The spatial dynamics of mitosis show a rapid and high mitotic increase in the distal regions but not in the proximal regions. Later, the proliferative zone shifts in sucessive mitotic waves to more proximal regions, paralleling the formation of the head ganglia and nerve cords.These results do not agree with theories of blastema formation through neoblast migration or through cell dedifferentiation, in as much as the local proliferation of neoblasts encountered is sufficiently high and early to account for the number of blastema cells found during period of regeneration. Therefore, we suggest that blastema formation during planarian regeneration occurs mainly through local neoblast proliferation.These results and the data obtained on regeneration rates enable us to suggest that nervous tissue may be one of the factors responsible for the differing mitotic increases found. Since neoblasts are the only planarian cells capable of mitosis, we suggest that neoblast proliferation could be under nervous tissue control through some stimulatory substance (s) released from the nerve terminals. The implications of this hypothesis for the maintenance of axial polarity are also discussed.

The stimulation of synaptosomal adenyl cyclase activity by the supernatant from rat brain cerebral cortex.

Effects of the 105000g supernatant on adenyl cyclase of rat cerebral cortex have been investigated and following results were obtained. 1) The 105000g supernatant significantly stimulated the activity of the synaptosomal adenyl cyclase in rat cerebral cortex. As the concentration of the supernatant increased, the progressive stimulation was observed with a plateau at a concentration of about 200 μg of protein. 2) This stimulatory effect of the supernatant was completely abolished by a dialysis of the supernatant for 24 hours at 4°. 3) When the enzyme system was incubated with combination of the supernatant and norepinephrine (100 μM), no activation by norepinephrine was observed. 4) Studies on the combined effects of the supernatant and sodium fluoride (NaF) showed that the supernatant additively enhanced NaF stimulated adenyl cyclase activity. 5) Gel filtration indicated that this stimulatory substance (s) in the supernatant had a molecular weight of 1000-1300.

Purification and regulatory properties of chicken heart prostaglandin E 9-ketoreductase.

Prostaglandin E 9-ketoreductase was purified from chicken heart by ammonium sulfate fractionation, and DEAE-Sephadex, hydroxylapatite and phosphocellulose chromatography. Two peaks of activity were resolved during the phosphocellulose chromatographic step. Both peaks were stimulated by a substance that was not bound to the phosphocellulose column. This stimulatory substance was destroyed by treatment with phosphodiesterase and 0.1 M NaOH. It was heat-stable (100 degrees, 2 min), nondialyzable, and resistant to treatment with pronase, ribonuclease, and deoxyribonuclease; but it was dialyzable after heating or digestion with pronase. Sodium pyrophosphate also enhanced the activities of the prostaglandin E 9-ketoreductases as did angiotensin I; but not angiotensin II. In the presence of 3':5'-cyclic AMP, AMP, or several other ribonucleotides, the enhancing effects of the natural stimulatory substance, sodium pyrophosphate or angiotensin I were blocked, but these ribonucleotides themselves had little effect on the enzymes activity. The substrate specificities of the two prostaglandin E 9-ketoreductases were also studied. Both the 9-keto group and the 15-keto group of 15-ketoprostaglandin F2 alpha could be converted to the corresponding hydroxyl group; the 15-keto group was reduced faster than the 9-keto group. Prostaglandin D2, a prostaglandin with a 9-hydroxyl and an 11-keto group, could not be converted to prostaglandin F2 alpha nor could cyclohexanone be converted to cyclohexanol by the prostaglandin E 9-ketoreductase.

Effects of Various Agents on Synaptosomal Adenylate Cyclase Activity in the Absence and Presence of the Boiled Supernatant

A study was made of the effects of various agents on adenylate cyclase in synaptosomes in both the absence and presence of the boiled supernatant from rat cerebral cortex. The activity of adenylate cyclase in these preparations was inhibited by the sulfhydryl reactive agent p-chloromercuribenzoate. Sulfhydryl compounds such cysteine, glutathione and Coenzyme A stimulated the enzymic activity in both the absence and presence of the boiled supernatant. The chelating agent 1.2-bis-(2-dicarboxymethylaminoethoxy)ethane caused a stimulation of the enzymic activity with and without the presence of the boiled supernatant. Adenine nucleotide (adenine, adenosine, AMP and ADP), GTP, Pi and carbamylcholine seemed to have little effect. The stimulatory substance in the boiled supernatant was estimated to have a molecular weight in the range of 1,000–1,300.

Production of colony-stimulating activity by human lymphocytes.

THE clonal growth of human bone marrow cells in vitro requires the presence of a stimulatory substance(s) designated colony-stimulating activity (CSA)1,2. Data available from various clinical and animal experiments suggest that CSA is, or is related to, a physiological controller of granulopoiesis3,4. Although the precise biological role of CSA is still uncertain, considerable insights into leukopoietic mechanisms have been gained from the study of interactions of CSA and haemopoietic cells in vitro. Two sources of CSA have been identified that stimulate colony formation from human bone marrow: certain foetal tissues and peripheral blood leucocytes3,4. The observation that CSA is produced by white cells led naturally to hypotheses concerning the feedback control of stem cells by mature leukocytes3,5.

Isolation and Identification of Stimulatory Substance Involved in the Associative Growth of Cheese Cultures

The stimulatory material produced in milk by two strains of Streptococcus cremoris (KH and HC) which enhanced the rate of acid production by S. cremoris R6 in milk has been identified as guanine by ultraviolet spectral analysis and other properties. Pure preparations of purines like adenine, guanine, and guanosine behaved similarly and enchanced acid production and proteolysis in milk by S. cremoris R6.

The effect of pineal extracts on hormone-induced rat uterine contractility.

Crude pineal extracts are apparently capable of exerting both a stimulatory and an inhibitory effect on the reproductive system of laboratory animals. The two active influences were separated as far as possible by means of TCA extraction (Reiss, Davis, Sideman, Mauer & Plichta, 1963) and the general methods of preparing the various cattle pineal fractions, used in the present study, were described. The inhibitory activity was found mainly in the supernatant fluid while the precipitate contained a stimulatory substance. The present paper reports on the activity of a few of these pineal fractions on rat uterine contractility induced in vitro by acetylcholine, pitocin and serotonin. Melatonin inhibited serotonin-induced rat uterine contractions (Hertz-Eshel & Rahamimoff, 1965) as well as pitocin-induced uterine contractions of women and mice (Davis, McGowan & Uroskie, in preparation). Oxytocic activity was isolated from bovine pineal gland (Milcu, Pavel & Neacsu, 1963). The effect of pineal amines on various types of mammalian smooth muscle has received consideration (Wurtman, Axelrod & Kelly, 1968). Uteri from adult Sprague Dawley rats (250 g) were suspended in 30 ml aerated de Jalon solution at 29-5° C and a single uterine contraction was induced by administering either one of three standards : 2 μg acetylcholine, 10 mu pitocin or 2 μg ofserotonin. The pineal extract was given in combination with the standard between two standard-induced contractions. The freezedried bovine pineal powders used were: TCA extract (Dowex, pH 7-4) ; super¬ natant of the original boiled residue from a simple aqueous extract; aqueous supernatant in a dialysing bag. Study 1 (Text-fig. 1). Trichloroacetic acid extract (0-1, 0-5 or 1-0 mg) was injected into the muscle bath simultaneously with serotonin (2 μg) or pitocin (10 mu) and exposed to the uterine horn for 3 min. For comparison, serotonin

The structure of leucogenenol.

The structure of leucogenenol, a blood cell stimulatory substance has now been determined. The compound forms a number of salts, a monomethyl enol ether, a derived tetra-acetate, and a hexabenzoate. Leucogenenol is hydrolysed to form 1,2-dihydroxy-3-methyl-5-oxocyclohexanecarboxylic acid, 3-hydroxy-3-hydroxymethyl-5-methylcyclohexane-1,2-dione, glycolaldehyde, aminoacetaldehyde and ammonia. This evidence and spectroscopic data indicate that leucogenenol is 2-(1,2-dihydroxy-3-methyl-5-oxocyclohexyl)-3,11-dihydroxy-11-(hydroxymethyl)-9-methyl-1-oxa-5-azaspiro[5,5]undeca-2,4-dien-7-one.

The role of aqueous humor, insulin and trivalent chromium in glucose utilization of rat lenses

Aqueous humor contains a stimulatory factor to lens glucose utilization not found in artificial media. Systemic insulin administration, in the presence of trace quantities of trivalent chromium, further enhances the stimulatory effect of aqueous humor. Excess amounts of trivalent chromium partially reverse the inttinsic stimulation, while in vivo injection of insulin overcomes the chromium inhibition. It is not known whether or not the stimulatory substance is insulin itself. It is believed, however, that trivalent chromium ion and the stimulator act in the area of the lens capsule.

The release of adenosine triphosphate from frog skeletal muscle in vitro.

1. Active frog sartorius muscle in vitro liberates a substance into the bathing solution which has a pronounced stimulatory action on the frog heart.2. The stimulatory effect is not due to an increase in the K(+) concentration of the bathing solution, nor is it due to the liberation of catecholamines.3. In a molecular sieve chromatography procedure the stimulatory substance can be eluted in a single fraction which shows a maximum absorption of U.V. light at a wave-length of 265 nm, indicative of the presence of substances containing a purine ring.4. Low concentrations (10(-7)-10(-8) g/ml.) of adenosine triphosphate (ATP), adenosine diphosphate (ADP) and uridine triphosphate (UTP) have a marked stimulatory action on the frog heart. The action of ATP and ADP on the heart is qualitatively very similar to that of the muscle bathing solution, while the action of UTP is distinctly different. The triphosphates of inosine, cytidine and guanosine stimulate the heart when in high concentration only. Adenosine and adenosine monophosphate do not stimulate the heart.5. Incubation of the muscle bathing solution and of solutions of ATP with the enzyme apyrase for the same time produces a similar marked reduction in the stimulatory action of both on the heart. Apyrase catalyses the break-down of nucleotide triphosphates to monophosphates.6. The elution behaviour of the stimulatory substance determined by molecular sieve chromatography is the same as that for ATP.7. The muscle bathing solution causes light to be emitted from firefly lantern extract, the pattern of light emission being similar to that produced by nucleotide triphosphates.8. The concentrations of ATP having the same quantitative action on the frog heart and on firefly extract as a given muscle bathing solution are almost identical, whereas the matching concentrations of ADP and UTP in the two methods of assay are widely different.9. It is concluded that ATP is released from active frog skeletal muscle in vitro. This release may play an important part in the reactive hyperaemia of muscular exercise since ATP has a powerful vasodilator action.

Studies on the production of perithecial stromata by Cordyceps militaris in artificial culture

Some nutritional and environmental factors which influence the production and maturation of perithecial stromata of Cordyceps militaris (Linnaeus) Link in artificial culture have been studied. The fungus grows vegetatively in defined media with a variety of carbon and nitrogen sources without added vitamins, and can use nitrate as sole nitrogen source, but forms stromata only under special nutritional conditions such as are provided by a medium of sterilized soaked rice grains. Illumination at more than 3 foot-candles is necessary for stromata to be initiated, and an intensity greater than a threshold value between 15 and 90 ft-c is necessary for production of mature perithecia. Temperatures above about 22 °C depress stroma development. So, too, does excessive aeration, which removes a gaseous or volatile stimulatory substance which is produced by the cultures. Media found suitable for stroma production include mineral salt solutions with either starch or sucrose associated with either haemoglobin or casein or peptone. Amino acids, used singly or in mixture, and glucose were not favorable. There was no evidence that fruiting required growth factors. The results support the view that stroma production is favored by media with carbon and nitrogen sources which cannot be assimilated until they have been hydrolyzed, and which consequently sustain favorably low concentrations of assimilable nutrients over a long period.

The stimulatory effect of Aureobasidium pullulans on rhizomorph development of Armillaria mellea in autoclaved soil.

Armillaria mellea (Fr.) Quél, was able to develop an extensive rhizomorph system in autoclaved soil in two-section petri plates if the stimulatory substance produced by Aureobasidium pullulans (de Bary) Arnaud was present. A sufficient quantity of the substance was produced by A. pullulans on a glucose–asparagine agar substrate but not on an autoclaved soil substrate.

Factors Affecting Growth of Streptococcus agalactiae in Milk

Factors that might affect the growth of Streptococcus agalactiae in milk were studied. Raw or pasteurized milk was serially diluted in steamed milk (100C, 30min) and acid production used to measure the growth of S. agalactiae 660 by determining the pH of the samples after incubation at 37C for 24 hr. The inhibitory titer, represented by the reciprocal of the highest dilution of milk showing maximum inhibition of acid production, was lowered when the milk used for diluent was heated for 30min at 80 or 90C, for 10min at 100C, or 15min at 121C, and when the incubation period of the test was increased to 48 hr. The inhibitory titer was progressively increased when 5 to 30μg of ammonium thiocyanate per ml of milk was added to pasteurized milk and the diluent. A stimulatory substance that counteracted the inhibitory activity of milk for S. agalactiae could be removed from whey by dialysis and ultrafiltration. Based on a comparison of the inhibitory activity curves, 20 colostrum samples tested had more stimulatory substance but less lactoperoxidase than milk. The inactivation of the inhibitory activity of milk by cysteine, glutathione, catalase, and horse-radish peroxidase confirmed the work of others who used organisms other than S. agalactiae.

Increased acid secretion from Heidenhain pouches by shunting colonic venous blood around the liver.

CREATION of a portacaval shunt in the dog is followed by increased secretion of acid from an indicator Heidenhain pouch.1One explanation for this finding is that a substance is released into the portal blood which has the capacity to stimulate gastric secretion, that it is normally inactivated in the liver, and that its effect becomes prominent when such inactivation is diminished due to diversion of portal blood around the liver. If this explanation is correct, it is of interest to determine where in the portal bed this stimulatory substance originates. We have presented experiments using selective shunting of portal blood which show it originates in the intestine distal to the midportion of the duodenum.2The present experiments were designed to determine whether it originates in the colon or small bowel or both. Methods Heidenhain pouches were fashioned from the greater curvature of the stomach in six mongrel

STIMULATION OF RHIZOMORPH DEVELOPMENT OF ARMILLARIA MELLEA BY AUREOBASIDIUM PULLULANS IN ARTIFICIAL CULTURE

In artificial culture, Aureobasidium pullulans (de Bary) Arnaud produced a stimulatory effect on rhizomorph development of Armillaria mellea (Fr.) Quél. Tests were carried out on malt agar, potato dextrose agar, and a chemically defined synthetic medium. The stimulatory substance (or substances) produced by A. pullulans was extracellular, diffusible in the medium and volatile. Tests indicated that the stimulation was caused by something other than ethanol or indoleacetic acid.