Identification of the main generator source of longitudinal muscle contraction in the earthworm ventral nerve cord

The main generator source of a longitudinal muscle contraction was identified as an M (mechanical-stimulus-sensitive) circuit composed of a presynaptic M-1 neuron and a postsynaptic M-2 neuron in the ventral nerve cord of the earthworm, Amynthas hawayanus, by simultaneous intracellular response recording and Lucifer Yellow-CH injection with two microelectrodes. Five-peaked responses were evoked in both neurons by a mechanical, but not by an electrical, stimulus to the mechanoreceptor in the shaft of a seta at the opposite side of an epidermis-muscle-nerve-cord preparation. This response was correlated to 84% of the amplitude, 73% of the rising rate and 81% of the duration of a longitudinal muscle contraction recorded by a mechano-electrical transducer after eliminating the other possible generator sources by partitioning the epidermis-muscle piece of this preparation. The pre- and postsynaptic relationship between these two neurons was determined by alternately stimulating and recording with two microelectrodes. Images of the Lucifer Yellow-CH-filled M-1 and M-2 neurons showed that both of them are composed of bundles of longitudinal processes situated on the side of the nerve cord opposite to stimulation. The M-1 neuron has an afferent process (A1) in the first nerve at the stimulated side of this preparation and the M-2 neuron has two efferent processes (E1 and E3) in the first and third nerves at the recording side where their effector muscle cell was identified by a third microelectrode.

Facilitation of the main generator source of earthworm muscle contraction by a peripheral neuron

A constant facilitation of responses evoked in the earthworm muscle contraction generator neurons by responses evoked in the neurons of its peripheral nervous system was demonstrated. It is based on the proposal that these two responses are bifurcations of an afferent response evoked by the same peripheral mechanical stimulus but converging again on this central neuron. A single-peaked generator response without facilitation was demonstrated by sectioning the afferent route of the peripheral facilitatory modulatory response, or conditioning response (CR). The multipeaked response could be restored by restimulating the sectioned modulatory neuron with an intracellular substitutive conditioning stimulus (SCS). These multi-peaked responses were proposed to be the result of reverberating the original single peaked unconditioned response (UR) through a parallel (P) neuronal circuit which receives the facilitation of the peripheral modulatory neuron. This peripheral modulatory neuron was named "Peri-Kästchen" (PK) neuron because it has about 20 peripheral processes distributed on the surface of a Kästchen of longitudinal muscle cells on the body wall of this preparation as revealed by the Lucifer Yellow-CH-filling method.

Persistent attenuation and enhancement of the earthworm main muscle contraction generator response induced by repeated stimulation of a peripheral neuron

Responses evoked in the earthworm, Amynthas hawayanus, main muscle contraction generator M-2 (postsynaptic mechanical-stimulus-sensitive) neuron by threshold mechanical stimuli in 2-s intertrial intervals (ITI) were used as the control or unconditioned responses (UR). Their attenuation induced by decreasing these intervals in non-associative conditioning and their enhancement induced by associating the unconditioned stimuli (US) to a train of short (0.1 s) hyperpolarizing electrical substitutive conditioning stimuli (SCS) in the Peri-Kästchen (PK) neuron were measured in four parameters, i.e., peak numbers (N) and amplitude ()averaged from 120 responses, sum of these amplitudes (SAMP) and the highest peak amplitude (V) over a period of 4 min. Persistent attenuation similar to habituation was induced by decreasing the control ITI to 0.5 s and 2.0 s in non-associative conditioning within less than 4 min. Dishabituation was induced by randomly pairing one of these habituated US to an electrical stimulus in the PK neuron. All four parameters of the UR were enhanced by forward (SCS-US), but not backward (US-SCS), association of the US with 25, 100 and 250-Hz trains of SCS with 40-ms interstimulus intervals (ISI) for 4 min and persisted for another 4 min after turning off the SCS. The enhancement of these parameters was proportional to the SCS frequencies in the train. No UR was evoked by the SCS when the US was turned off after 4 min of classical conditioning.

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.

Radio-frequency-based measurement methods for bearing current analysis in induction motors

The most common reason for a low-voltage induction motor breakdown is a bearing failure. Along with the increasing popularity of modern frequency converters, bearing failures have become the most important motor fault type. Conditions in which bearing currents are likely to occur are generated as a side effect of fast du/dt switching transients. Once present, different types of bearing currents can accelerate the mechanical wear of bearings by causing deformation of metal parts in the bearing and degradation of the lubricating oil properties.The bearing current phenomena are well known, and several bearing current measurement and mitigation methods have been proposed. Nevertheless, in order to develop more feasible methods to measure and mitigate bearing currents, better knowledge of the phenomena is required. When mechanical wear is caused by bearing currents, the resulting aging impact has to be monitored and dealt with. Moreover, because of the stepwise aging mechanism, periodically executed condition monitoring measurements have been found ineffective. Thus, there is a need for feasible bearing current measurement methods that can be applied in parallel with the normal operation of series production drive systems. In order to reach the objectives of feasibility and applicability, nonintrusive measurement methods are preferred. In this doctoral dissertation, the characteristics and conditions of bearings that are related to the occurrence of different kinds of bearing currents are studied. Further, the study introduces some nonintrusive radio-frequency-signal-based approaches to detect and measure parameters that are associated with the accelerated bearing wear caused by bearing currents.

Study of Control Methods of a High-Frequency Cycloconverter for Domestic Induction Heating Appliances

A high-frequency cyclonverter acts as a direct ac-to-ac power converter circuit that does not require a diode bidge rectifier. Bridgeless topology makes it possible to remove forward voltage drop losses that are present in a diode bridge. In addition, the on-state losses can be reduced to 1.5 times the on-state resistance of switches in half-bridge operation of the cycloconverter. A high-frequency cycloconverter is reviewed and the charging effect of the dc-capacitors in ``back-to-back'' or synchronous mode operation operation is analyzed. In addition, a control method is introduced for regulating dc-voltage of the ac-side capacitors in synchronous operation mode. The controller regulates the dc-capacitors and prevents switches from reaching overvoltage level. This can be accomplished by variating phase-shift between the upper and the lower gate signals. By adding phase-shift between the gate signal pairs, the charge stored in the energy storage capacitors can be discharged through the resonant load and substantially, the output resonant current amplitude can be improved. The above goals are analyzed and illustrated with simulation. Theory is supported with practical measurements where the proposed control method is implemented in an FPGA device and tested with a high-frequency cycloconverter using super-junction power MOSFETs as switching devices.

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.

Effect of dietary (n-3) highly unsaturated fatty acids on growth and survival of fat snook (Centropomus parallelus, Pisces: Centropomidae) larvae during first feeding

The effect of rotifers, Brachionus rotundiformis (S-type), fed three different diets: A (rotifer fed Nannochloropsis oculata), B (rotifer fed N. oculata and baker's yeast, 1:1), and C (rotifer fed N. oculata and baker's yeast, 1:1, and enriched with Selcoâ), was evaluated based on the survival, growth and swim bladder inflation rate of fat snook larvae. Rotifers of treatment A had higher levels (4.58 mg/g dry weight) of eicosapentaenoic acid (EPA) than B (1.81 mg/g dry weight), and similar levels (0.04 and 0.06 mg/g dry weight, respectively) of docosahexaenoic acid (DHA). Rotifers of treatment C had the highest levels of EPA (13.2 mg/g dry weight) and DHA (6.08 mg/g dry weight). Fat snook eggs were obtained by spawning induction with human chorionic gonadotropin. Thirty hours after hatching, 30 larvae/liter were stocked in black cylindric-conical tanks (36-liter capacity). After 14 days of culture, there were no significant differences among treatments. Mean standard length was 3.13 mm for treatment A, 3.17 mm for B, and 3.39 mm for C. Mean survival rates were very low (2.7% for treatment A, 2.3% for B, and 1.8% for C). Swim bladder inflation rates were 34.7% for treatment A, 27.1% for B, and 11.9% for C. The lack of differences in growth and survival among treatments showed that the improvement of the dietary value of rotifer may not have been sufficient to solve the problem of larval rearing. Some other factor, probably pertaining to the quality of the larvae, may have negatively influenced survival.

Influences of alpha-tocopherol on cholesterol metabolism and fatty streak development in apolipoprotein E-deficient mice fed an atherogenic diet

Although the role of oxidized lipoproteins is well known in atherogenesis, the role of vitamin E supplementation is still controversial. There is also little information about cholesterol metabolism (hepatic concentration and fecal excretion) in the new models of atherosclerosis. In the present study, we evaluated the effect of moderate vitamin E supplementation on cholesterol metabolism and atherogenesis in apolipoprotein E (apo E)-deficient mice. Apo E-deficient mice were fed an atherogenic diet containing 40 or 400 mg/kg of alpha-tocopherol acetate for 6 weeks. Total cholesterol in serum and liver and 3-OH-alpha-sterols in feces, and fecal excretion of bile acids were determined and histological analyses of aortic lesion were performed. A vitamin E-rich diet did not affect body weight, food intake or serum cholesterol. Serum and hepatic concentrations of cholesterol as well as sterol concentration in feces were similar in both groups. However, when compared to controls, the alpha-tocopherol-treated mice showed a reduction of about 60% in the atherosclerotic lesions when both the sum of lesion areas and the average of the largest lesion area were considered. These results demonstrate that supplementation of moderate doses of alpha-tocopherol was able to slow atherogenesis in apo E-deficient mice and to reduce atherogenic lipoproteins without modifying the hepatic pool or fecal excretion of cholesterol and bile acids.

Induction of interleukin-10 by HIV antigens in peripheral mononuclear cells of health care workers after occupational exposure to HIV-1-positive blood

Evaluation of HIV-induced IL-2 production by peripheral blood mononuclear cells (PBMC) and HIV-specific T helper and cytotoxic T lymphocyte (CTL) responses in health care workers (HCW) occupationally exposed to HIV reveals a high rate of response to HIV among non-seroconverters. IL-10 is also known to interfere with HIV infection in vitro. To evaluate the induction of IL-10 by HIV antigens in HCW occupationally exposed to HIV, 18 HCW with percutaneous injury were enrolled in this study, 9 of them exposed to HIV-contaminated blood, and 9 exposed to HIV-negative blood. PBMC were incubated on plates coated with HIV-1 antigens, and IL-10 was measured in supernatants by ELISA. Five of nine HCW exposed to HIV-contaminated blood presented HIV-induced IL-10. Two of nine HCW exposed to HIV-negative source patients also had detectable levels of HIV-induced IL-10, one of them in the sample obtained on the day of accidental exposure. There was a relationship between the type of device involved in injury and IL-10 production. Individuals exposed to hollow needles or scalpels presented HIV-induced IL-10, whereas those exposed to solid needles and to digital puncture did not, suggesting a relationship between infectious load and IL-10. Although occupational exposure to HIV leads to a low rate of seroconversion, these individuals can develop an antigen-specific immune response characterized in our study by induction of IL-10 in PBMC in vitro.

Induction of liver monooxygenases by annatto and bixin in female rats

Annatto or urucum is an orange-yellow dye obtained from Bixa orellana seeds. It has been used as a natural dye in a variety of food products, drugs and cosmetics, and also in Brazilian cuisine as a condiment ('colorau'). Bixin, a carotenoid devoid of provitamin A activity, is the main pigment found in annatto. Some carotenoids (canthaxanthin, astaxanthin and ß-Apo-8'-carotenal) are known to be potent inducers of CYP1A1, a property not shared by others (ß-carotene, lycopene and lutein). Little is known, however, about the CYP1A1-inducing properties of bixin and annatto. The present study was performed to determine the effects of an annatto extract (28% bixin) and bixin (95% pure) on rat liver monooxygenases. Adult female Wistar rats were treated by gavage with daily doses of annatto (250 mg/kg body weight, which contains approximately 70 mg bixin/kg body weight), bixin (250 mg/kg body weight) or the vehicle only (corn oil, 3.75 g/kg body weight) for 5 consecutive days, or were not treated (untreated control). The activities of aniline-4-hydroxylase (A4H), ethoxycoumarin-O-deethylase (ECOD), ethoxy- (EROD), methoxy- (MROD), pentoxy- (PROD) and benzyloxy- (BROD) resorufin-O-dealkylases were measured in liver microsomes. Annatto (250 mg/kg containing 70 mg bixin/kg) induced EROD (3.8x), MROD (4.2x), BROD (3.3x) and PROD (2.4x). Bixin (250 mg/kg) was a weaker inducer of EROD (2.7x), MROD (2.3x) and BROD (1.9x) and did not alter PROD, A4H or ECOD activities. These results suggest that constituents of the extract other than bixin play an important role in the induction of CYP1A and CYP2B observed with annatto food colorings.