Callus induction and plant regeneration by Brazilian triticale and wheat genotypes

In order to determine the in vitro behavior of Brazilian triticale, 16 triticale genotypes, and three wheat genotypes used as checks, were sown in June 1994. The explants used were immature embryos. In addition to the genotype tests, two culture media for callus induction were also evaluated, i.e., MS (Murashige and Skoog, Physiol. Plant. 15: 473-497, 1962) medium containing 2.0 mg 2,4D/l, and MS medium containing 4.0 mg 2,4D/l. The plant regeneration protocol used was the one employed at the Laboratório de Cultura de Tecidos, Departamento de Plantas de Lavoura, Universidade Federal do Rio Grande do Sul, for wheat. Differences in plant regeneration were observed both among triticale and wheat genotypes, with triticale usually showing better regeneration than wheat. No differences were observed between the callus induction media.

Induction of mutations for earliness in the soybean cultivar Paraná

Mutation induction breeding through gamma ray seed treatment (22 krad) was used to obtain earliness in the soybean cultivar Paraná. Plants flowering earlier than Paraná were selected at the M3 generation. Eight mutant lines were isolated by further selections through several generations and tested in four yield trials carried out in 85/86 (Piracicaba) and 86/87 (Piracicaba, Sertãozinho and Assis) in São Paulo State. The following data were taken in these trials: number of days to flowering, yield, total plant height and height to pod insertion, flower color, pod pubescence and seed and plant growth type. Two of the mutants are of agronomic interest because they were, on average, seven to 10 days earlier in maturity than the control in all trials. Except for shorter plant height, the other agronomic characteristics remained unaltered.

Prolactin inhibits auto- and cross-induction of thyroid hormone and estrogen receptor and vitellogenin genes in adult Xenopus (Amphibia) hepatocytes

It is well known that virtually every tissue of the amphibian larvae is highly sensitive to the mutually antagonistic actions of thyroid hormone (TH) and prolactin (PRL), but it is not known if adult amphibian tissues respond similarly to these two hormones. We have previously shown that very low doses of triiodothyronine (T3) rapidly and strongly potentiate the activation of silent vitellogenin (Vit) genes by estrogen (E2) and the autoinduction of estrogen receptor (ER) transcripts in primary cultures of adult Xenopus hepatocytes. This response to T3 is accompanied by the upregulation of thyroid hormone receptor b (TRb) mRNA. Using Northern blot and RNase protection assays, we now show that ovine PRL added for 12 h along with 2 x 10-9 M T3 will completely prevent potentiation of E2 induction of Vit mRNA in primary cultures of adult Xenopus hepatocytes. PRL also abolished the auto-upregulation of TRb mRNA and the cross-activation of autoinduction of ER mRNA. Thus, we show for the first time that the anti-TH action of PRL that is manifested in Xenopus tadpole tissues during metamorphosis is retained in adult liver, and suggest that the mutually antagonistic actions of the two hormones may be brought about by similar molecular mechanisms in larval and adult amphibian tissues

Volatile cardioplegia: fast normothermic cardiac arrest induction and recovery with halothane in isolated rat hearts

To study the effect of halothane as a cardioplegic agent, ten Wistar rats were anesthetized by ether inhalation and their hearts were perfused in a Langendorff system with Krebs-Henseleit solution (36oC; 90 cm H2O pressure). After a 15-min period for stabilization the control values for heart rate, force (T), dT/dt and coronary flow were recorded and a halothane-enriched solution (same temperature and pressure) was perfused until cardiac arrest was obtained. The same Krebs-Henseleit solution was reperfused again and the parameters studied were recorded after 1, 3, 5, 10, 20 and 30 min. Cardiac arrest occurred in all hearts during the first two min of perfusion with halothane-bubbled solution. One minute after reperfusion without halothane, the following parameters reported in terms of control values were obtained: 90.5% of control heart rate (266.9 ± 43.4 to 231.5 ± 71.0 bpm), 20.2% of the force (1.83 ± 0.28 to 0.37 ± 0.25 g), 19.8% of dT/dt (46.0 ± 7.0 to 9.3 ± 6.0 g/s) and 90.8% of coronary flow (9.9 ± 1.5 to 9.4 ± 1.5 ml/min). After 3 min of perfusion they changed to 99.0% heart rate (261.0 ± 48.2), 98.9% force (1.81 ± 0.33), 98.6 dT/dt (45.0 ± 8.2) and 94.8% coronary flow (9.3 ± 1.4). At 5 min 100.8% (267.0 ± 40.6) heart rate, 105.0% (1.92 ± 0.29) force and 104.4% (48.2 ± 7.2) dT/dt were recorded and maintained without significant differences (P>0.01) until the end of the experiment. These data demonstrate that volatile cardioplegia with halothane is an effective technique for fast induction of and prompt recovery from normothermic cardiac arrest of the rat heart

Induction of neutralizing antibodies in mice immunized with scorpion toxins detoxified by liposomal entrapment

The possibility of producing neutralizing antibodies against the lethal effects of scorpion toxins was evaluated in the mouse model by immunization with an immunogen devoid of toxicity. A toxic fraction (5 mg) from the venom of the scorpion Tityus serrulatus was entrapped in sphingomyelin-cholesterol liposomes. The liposomes were treated for 1 h at 37oC with a 1% (w/w) trypsin solution in 0.2 M sodium carbonate buffer, pH 8.3. This treatment led to a strong reduction in venom toxicity. Immunization was performed as follows: mice were injected sc with 20 µg of the liposome-entrapped toxic fraction on days 1 and 21 and a final injection (20 µg) was administered ip on day 36. After injection of the immunogen, all mice developed an IgG response which was shown to be specific for the toxic antigen. The antibodies were measured 10 days after the end of the immunization protocol. In an in vitro neutralization assay we observed that pre-incubation of a lethal dose of the toxic fraction with immune serum strongly reduced its toxicity. In vivo protection assays showed that mice with anti-toxin antibodies could resist the challenge with the toxic fraction, which killed, 30 min after injection, all non-immune control mice

Role of mast cell- and non-mast cell-derived inflammatory mediators in immunologic induction of synaptic plasticity

We have previously discovered a long-lasting enhancement of synaptic transmission in mammal autonomic ganglia caused by immunological activation of ganglionic mast cells. Subsequent to mast cell activation, lipid and peptide mediators are released which may modulate synaptic function. In this study we determined whether some mast cell-derived mediators, prostaglandin D2 (PGD2; 1.0 µM), platelet aggregating factor (PAF; 0.3 µM) and U44619 (a thromboxane analogue; 1.0 µM), and also endothelin-1 (ET-1; 0.5 µM) induce synaptic potentiation in the guinea pig superior cervical ganglion (SCG), and compared their effects on synaptic transmission with those induced by a sensitizing antigen, ovalbumin (OVA; 10 µg/ml). The experiments were carried out on SCGs isolated from adult male guinea pigs (200-250 g) actively sensitized to OVA, maintained in oxygenated Locke solution at 37oC. Synaptic potentiation was measured through alterations of the integral of the post-ganglionic compound action potential (CAP). All agents tested caused long-term (LTP; duration ³30 min) or short-term (STP; <30 min) potentiation of synaptic efficacy, as measured by the increase in the integral of the post-ganglionic CAP. The magnitude of mediator-induced potentiation was never the same as the antigen-induced long-term potentiation (A-LTP). The agent that best mimicked the antigen was PGD2, which induced a 75% increase in CAP integral for LTP (antigen: 94%) and a 34% increase for STP (antigen: 91%). PAF-, U44619-, and ET-1-induced increases in CAP integral ranged for LTP from 34 to 47%, and for STP from 0 to 26%. These results suggest that the agents investigated may participate in the induction of A-LTP

Induction of cell-mediated immunity during early stages of infection with intracellular protozoa

Toxoplasma gondii and Trypanosoma cruzi are intracellular parasites which, as part of their life cycle, induce a potent cell-mediated immunity (CMI) maintained by Th1 lymphocytes and IFN-g. In both cases, induction of a strong CMI is thought to protect the host against rapid parasite multiplication and consequent pathology and lethality during the acute phase of infection. However, the parasitic infection is not eliminated by the immune system and the vertebrate host serves as a parasite reservoir. In contrast, Leishmania sp, which is a slow growing parasite, appears to evade induction of CMI during early stages of infection as a strategy for surviving in a hostile environment (i.e., inside the macrophages which are their obligatory niche in the vertebrate host). Recent reports show that the initiation of IL-12 synthesis by macrophages during these parasitic infections is a key event in regulating CMI and disease outcome. The studies reviewed here indicate that activation/inhibition of distinct signaling pathways and certain macrophage functions by intracellular protozoa are important events in inducing/modulating the immune response of their vertebrate hosts, allowing parasite and host survival and therefore maintaining parasite life cycles.

Oral tolerance induction with altered forms of ovalbumin

As a T cell-dependent phenomenon, oral tolerance is not expected to depend necessarily on native configuration of antigens. We investigated the induction of oral tolerance with modified ovalbumin (Ova). Oral administration of heat-denatured (HD-Ova) and cyanogen bromide-degraded ovalbumin was less effective than native Ova in inducing oral tolerance in B6D2F1 mice. HD-Ova was effective in suppressing delayed-type hypersensitivity (DTH) reactions but did not suppress specific antibody formation. Injection of Ova directly into the stomach, but not into the ileum or cecum, suppressed subsequent immunization to DTH reactions. Gavage with protease inhibitors (aprotinin or ovomucoid) before gavage with Ova was ineffective in blocking tolerance induction. Treatment with hydroxyurea to destroy cycling cells 24 h before gavage with Ova blocked oral tolerance induction and also the possibility to passively transfer tolerance to naive recipients with the serum of mice gavaged with Ova 1 h before. The implications of these findings about oral tolerance induction are discussed

Effect of prostaglandin A1 in the induction of stress proteins in Aedes albopictus cells

Prostaglandins are natural fatty acid derivatives with diverse physiological effects, including immune function and the control of cell growth. While the action of prostaglandins in the induction of stress proteins in vertebrate cells is well documented, their functions in invertebrate cells have been poorly investigated. The purpose of the present study was to investigate the effect of prostaglandin A1 (PGA1; 0.25, 1.25 and 12.5 µg/ml) on protein synthesis during the growth of Aedes albopictus cells. We found that PGA1 stimulates the synthesis of several polypeptides with molecular masses of 87, 80, 70, 57, 29, 27 and 23 kDa in Aedes albopictus cells. When the proteins induced by PGA1 and those induced by heat treatment were compared by polyacrylamide gel electrophoresis, PGA1 was found to induce the stress proteins. The HSP70 family and the low-molecular weight polypeptides (29 and 27 kDa, respectively) were induced by PGA1 in the lag phase. We also observed that PGA1 is able to induce a 23-kDa polypeptide independently of the growth phase of the cell

Coupling of palmitate to ovalbumin inhibits the induction of oral tolerance

Oral tolerance is a phenomenon that may occur in animals exposed to protein antigens for the first time by the oral route. They become unable to produce immune responses at the levels normally observed when they are immunized parenterally with antigen in the presence of adjuvants. Lipids have been used as adjuvants for both parenteral and oral immunization. In the present study we coupled ovalbumin with palmitate residues by incubating the protein with the N-hydroxysuccinimide palmitate ester and tested the preparation for its ability to induce oral tolerance. This was performed by giving 20 mg of antigen to mice by the oral route 7 days prior to parenteral immunization in the presence of Al(OH)3. Mice were bled one week after receiving a booster that was given 2 weeks after primary immunization. Specific antibodies were detected by ELISA. Despite the fact that the conjugates are as immunogenic as the unmodified protein when parenterally injected in mice, they failed to induce oral tolerance. This discrepancy could be explained by differences in the intestinal absorption of the two forms of the antigen. In fact, when compared to the non-conjugated ovalbumin, a fast and high absorption of the lipid-conjugated form of ovalbumin was observed by "sandwich" ELISA.

Induction of apoptosis in cancer cells by tumor necrosis factor and butyrolactone, an inhibitor of cyclin-dependent kinases

Induction of apoptosis by tumor necrosis factor (TNF) is modulated by changes in the expression and activity of several cell cycle regulatory proteins. We examined the effects of TNF (1-100 ng/ml) and butyrolactone I (100 µM), a specific inhibitor of cyclin-dependent kinases (CDK) with high selectivity for CDK-1 and CDK-2, on three different cancer cell lines: WEHI, L929 and HeLa S3. Both compounds blocked cell growth, but only TNF induced the common events of apoptosis, i.e., chromatin condensation and ladder pattern of DNA fragmentation in these cell lines. The TNF-induced apoptosis events were increased in the presence of butyrolactone. In vitro phosphorylation assays for exogenous histone H1 and endogenous retinoblastoma protein (pRb) in the total cell lysates showed that treatment with both TNF and butyrolactone inhibited the histone H1 kinase (WEHI, L929 and HeLa) and pRb kinase (WEHI) activities of CDKs, as compared with the controls. The role of proteases in the TNF and butyrolactone-induced apoptosis was evaluated by comparing the number and expression of polypeptides in the cell lysates by gel electrophoresis. TNF and butyrolactone treatment caused the disappearance of several cellular protein bands in the region between 40-200 kDa, and the 110- 90- and 50-kDa proteins were identified as the major substrates, whose degradation was remarkably increased by the treatments. Interestingly, the loss of several cellular protein bands was associated with the marked accumulation of two proteins apparently of 60 and 70 kDa, which may be cleavage products of one or more proteins. These findings link the decrease of cyclin-dependent kinase activities to the increase of protease activities within the growth arrest and apoptosis pathways induced by TNF.

Induction of Fos protein immunoreactivity by spinal cord contusion

The objective of the present study was to identify neurons in the central nervous system that respond to spinal contusion injury in the rat by monitoring the expression of the nuclear protein encoded by the c-fos gene, an activity-dependent gene, in spinal cord and brainstem regions. Rats were anesthetized with urethane and the injury was produced by dropping a 5-g weight from 20.0 cm onto the exposed dura at the T10-L1 vertebral level (contusion group). The spinal cord was exposed but not lesioned in anesthetized control animals (laminectomy group); intact animals were also subjected to anesthesia (intact control). Behavioral alterations were analyzed by Tarlov/Bohlman scores, 2 h after the procedures and the animals were then perfused for immunocytochemistry. The patterns of Fos-like immunoreactivity (FLI) which were site-specific, reproducible and correlated with spinal laminae that respond predominantly to noxious stimulation or injury: laminae I-II (outer substantia gelatinosa) and X and the nucleus of the intermediolateral cell column. At the brain stem level FLI was detected in the reticular formation, area postrema and solitary tract nucleus of lesioned animals. No Fos staining was detected by immunocytochemistry in the intact control group. However, detection of FLI in the group submitted to anesthesia and surgical procedures, although less intense than in the lesion group, indicated that microtraumas may occur which are not detected by the Tarlov/Bohlman scores. There is both a local and remote effect of a distal contusion on the spinal cord of rats, implicating sensory neurons and centers related to autonomic control in the reaction to this kind of injury.

Regulation of the kinin receptors after induction of myocardial infarction: a mini-review

It is well known that the responses to vasoactive kinin peptides are mediated through the activation of two receptors termed bradykinin receptor B1 (B1R) and B2 (B2R). The physiologically prominent B2R subtype has certainly been the subject of more intensive efforts in structure-function studies and physiological investigations. However, the B1R activated by a class of kinin metabolites has emerged as an important subject of investigation within the study of the kallikrein-kinin system (KKS). Its inducible character under stress and tissue injury is therefore a field of major interest. Although the KKS has been associated with cardiovascular regulation since its discovery at the beginning of the last century, less is known about the B1R and B2R regulation in cardiovascular diseases like hypertension, myocardial infarction (MI) and their complications. This mini-review will summarize our findings on B1R and B2R regulation after induction of MI using a rat model. We will develop the hypothesis that differences in the expression of these receptors may be associated with a dual pathway of the KKS in the complex mechanisms of myocardial remodeling.

Stabilization of serum antibody responses triggered by initial mucosal contact with the antigen independently of oral tolerance induction

Initial contacts with a T-dependent antigen by mucosal routes may result in oral tolerance, defined as the inhibition of specific antibody formation after subsequent parenteral immunizations with the same antigen. We describe here an additional and permanent consequence of these initial contacts, namely, the blockade of secondary-type responsiveness to subsequent parenteral contacts with the antigen. When repeatedly boosted ip with small doses (3 µg) of ovalbumin (OVA) (or lysozyme), primed B6D2F1 mice showed progressively higher antibody responses. In contrast, mice primed after a single oral exposure to the antigen, although repeatedly boosted, maintained their secondary antibody titers on a level which was inversely proportional to the dose of antigen in the oral pretreatment. This phenomenon also occurred in situations in which oral tolerance was not induced. For example, senile 70-week-old B6D2F1 mice pretreated with a single gavage of 20 mg OVA did not become tolerant, i.e., they formed the same secondary levels of anti-OVA antibodies as non-pretreated mice. However, after 4 weekly challenges with 3 µg OVA ip, orally pretreated mice maintained the same anti-OVA serum levels, whereas the levels of control mice increased sequentially. This "stabilizing" effect of mucosal exposure was dose dependent, occurred with different proteins and was triggered by single or multiple oral or nasal exposures to the antigen.

Fever induction pathways: evidence from responses to systemic or local cytokine formation

The immune and central nervous systems are functionally connected and interacting. The concept that the immune signaling to the brain which induces fever during infection and inflammation is mediated by circulating cytokines has been traditionally accepted. Administration of bacterial lipopolysaccharide (LPS) induces the appearance of a so-termed "cytokine cascade" in the circulation more or less concomitantly to the developing febrile response. Also, LPS-like fever can be induced by systemic administration of key cytokines (IL-1ß, TNF-alpha, and others). However, anti-cytokine strategies against IL-1ß or TNF-alpha along with systemic injections of LPS frequently lead to attenuation of the later stages of the febrile response but not of the initial phase of fever, indicating that cytokines are rather involved in the maintenance than in the early induction of fever. Within the last years experimental evidence has accumulated indicating the existence of neural transport pathways of immune signals to the brain. Because subdiaphragmatic vagotomy prevents or attenuates fever in response to intraperitoneal or intravenous injections of LPS, a role for vagal afferent nerve fibers in fever induction has been proposed. Also other sensory nerves may participate in the manifestation of febrile responses under certain experimental conditions. Thus, injection of a small dose of LPS into an artificial subcutaneous chamber results in fever and formation of cytokines within the inflamed tissue around the site of injection. This febrile response can be blocked in part by injection of a local anesthetic into the subcutaneous chamber, indicating a participation of cutaneous afferent nerve signals in the manifestation of fever in this model. In conclusion, humoral signals and an inflammatory stimulation of afferent sensory nerves can participate in the generation and maintenance of a febrile response.