Axillary bud development in pineapple nodal segments correlates with changes on cell cycle gene expression, hormone level, and sucrose and glutamate contents

During the process of lateral organ development after plant decapitation, cell division and differentiation occur in a balanced manner initiated by specific signaling, which triggers the reentrance into the cell cycle. Here, we investigated short-term variations in the content of some endogenous signals, such as auxin, cytokinins (Cks), and other mitogenic stimuli (sucrose and glutamate), which are likely correlated with the cell cycle reactivation in the axillary bud primordium of pineapple nodal segments. Transcript levels of cell cycle-associated genes, CycD2;1, and histone H2A were analyzed. Nodal segments containing the quiescent axillary meristem cells were cultivated in vitro during 24 h after the apex removal and de-rooting. From the moment of stem apex and root removal, decapitated nodal segment (DNS) explants showed a lower indol-3-acetic acid (IAA) concentration than control explants, and soon after, an increase of endogenous sucrose and iP-type Cks were detected. The decrease of IAA may be the primary signal for cell cycle control early in G1 phase, leading to the upregulation of CycD2;1 gene in the first h. Later, the iP-type Cks and sucrose could have triggered the progression to S-phase since there was an increase in H2A expression at the eighth h. DNS explants revealed substantial increase in Z-type Cks and glutamate from the 12th h, suggesting that these mitogens could also operate in promoting pineapple cell cycle progression. We emphasize that the use of non-synchronized tissue rather than synchronous cell suspension culture makes it more difficult to interpret the results of a dynamic cell division process. However, pineapple nodal segments cultivated in vitro may serve as an interesting model to shed light on apical dominance release and the reentrance of quiescent axillary meristem cells into the cell cycle.

Correlation between citric acid and nitrate metabolisms during CAM cycle in the atmospheric bromeliad Tillandsia pohliana

Crassulacean acid metabolism (CAM) confers crucial adaptations for plants living under frequent environmental stresses. A wide metabolic plasticity can be found among CAM species regarding the type of storage carbohydrate, organic acid accumulated at night and decarboxylating system. Consequently, many aspects of the CAM pathway control are still elusive while the impact of this photosynthetic adaptation on nitrogen metabolism has remained largely unexplored. In this study, we investigated a possible link between the CAM cycle and the nitrogen assimilation in the atmospheric bromeliad Tillandsia pohliana by simultaneously characterizing the diel changes in key enzyme activities and metabolite levels of both organic acid and nitrate metabolisms. The results revealed that T. pohliana performed a typical CAM cycle in which phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxykinase phosphorylation seemed to play a crucial role to avoid futile cycles of carboxylation and decarboxylation. Unlike all other bromeliads previously investigated, almost equimolar concentrations of malate and citrate were accumulated at night. Moreover, a marked nocturnal depletion in the starch reservoirs and an atypical pattern of nitrate reduction restricted to the nighttime were also observed. Since reduction and assimilation of nitrate requires a massive supply of reducing power and energy and considering that T. pohliana lives overexposed to the sunlight, we hypothesize that citrate decarboxylation might be an accessory mechanism to increase internal CO(2) concentration during the day while its biosynthesis could provide NADH and ATP for nocturnal assimilation of nitrate. Therefore, besides delivering photoprotection during the day, citrate might represent a key component connecting both CAM pathway and nitrogen metabolism in T. pohliana: a scenario that certainly deserves further study not only in this species but also in other CAM plants that nocturnally accumulate citrate. (C) 2010 Elsevier GmbH. All rights reserved.

Specific leaf areas of the tank bromeliad Guzmania monostachia perform distinct functions in response to water shortage

Leaves comprise most of the vegetative body of tank bromeliads and are usually subjected to strong longitudinal gradients. For instance, while the leaf base is in contact with the water accumulated in the tank, the more light-exposed middle and upper leaf sections have no direct access to this water reservoir. Therefore, the present study attempted to investigate whether different leaf portions of Guzmania monostachia, a tank-forming C(3)-CAM bromeliad, play distinct physiological roles in response to water shortage, which is a major abiotic constraint in the epiphytic habitat. Internal and external morphological features, relative water content, pigment composition and the degree of CAM expression were evaluated in basal, middle and apical leaf portions in order to allow the establishment of correlations between the structure and the functional importance of each leaf region. Results indicated that besides marked structural differences, a high level of functional specialization is also present along the leaves of this bromeliad. When the tank water was depleted, the abundant hydrenchyma of basal leaf portions was the main reservoir for maintaining a stable water status in the photosynthetic tissues of the apical region. In contrast, the CAM pathway was intensified specifically in the upper leaf section, which is in agreement with the presence of features more suitable for the occurrence of photosynthesis at this portion. Gas exchange data indicated that internal recycling of respiratory CO(2) accounted for virtually all nighttime acid accumulation, characterizing a typical CAM-idling pathway in the drought-exposed plants. Altogether, these data reveal a remarkable physiological complexity along the leaves of G. monostachia, which might be a key adaptation to the intermittent water supply of the epiphytic niche. (C) 2009 Elsevier GmbH. All rights reserved.

Postnatal fluoxetine treatment affects the development of serotonergic neurons in rats

The goal of the present study was to investigate morphological changes in the serotonergic neurons/terminals in the dorsal (DR) and median (MnR) raphe nuclei and on the hippocampal dentate gyrus (DG) in neonatal rats treated from the 1st to the 21st postnatal day with fluoxetine (10 mg/kg sc, daily) or drug vehicle (0 9% saline 1 ml/kg). The results show that postnatal chronic treatment with fluoxetine promoted. (1) a smaller body weight increase during the pre-weaning period; (2) smaller number of 5-HT neurons in the DR, (3) smaller 5-HT neuronal cell bodies (area, perimeter and diameter) in the DR and the MnR and (4) diminished serotonergic terminals in the DG. These data suggest that the development of the serotonergic system was impaired and that early exposure to fluoxetine damaged the morphology of 5-HT neurons in young adult rats While these findings are consistent with other work, more studies are needed to better clarify the effects of postnatal chronic treatment with fluoxetine on the serotonergic system and, consequently, on the functions modulated by serotonin (C) 2010 Elsevier Ireland Ltd All rights reserved

Morphine treatment during pregnancy modulates behavioral selection in lactating rats

Previous studies have demonstrated that treatment of postpartum female rats with morphine inhibits maternal behavior and stimulates foraging. Exposure to drugs of abuse may result in a progressive enhancement of their reinforcing effects. Puerperal treatment with morphine leads to reverse tolerance to this drug. The present study investigated whether repeated morphine treatment during late pregnancy may influence the effects of different morphine dosages on behavioral selection in lactating rats. Females were simultaneously exposed to pups and insects, and the choice between taking care of the pups and hunting insects was observed. Female Wistar rats were treated with morphine (3.5 mg/kg/day, subcutaneous [s.c.]) or saline for 5 days beginning on pregnancy day 17. On day 5 of lactation, animals were acutely challenged with morphine (0.5, 1.0, or 1.5 mg/kg, s.c.; MM0.5, MM1.0, and MM1.5 groups, respectively) or saline (MS group) and tested for predatory hunting and maternal behavior. Control groups were pretreated with saline and challenged with morphine (SM0.5, SM1.0, and SM1.5 groups) or saline (SS group). Animals treated with morphine during late pregnancy and acutely challenged with 1.0 mg/kg morphine (MM1.0 group) exhibited significantly decreased maternal behavior and enhanced hunting. This effect was not evident with the 0.5 mg/kg dose. The 1.5 mg/kg morphine dose decreased maternal behavior and increased hunting in both the MM1.5 group and in animals challenged with morphine after previous saline treatment (SM1.5 group). These results provide evidence of plasticity of the opioidergic role in behavioral selection during lactation. (C) 2010 Elsevier Inc. All rights reserved.

The periaqueductal gray and its potential role in maternal behavior inhibition in response to predatory threats

Animals faced with conflicting cues, such as predatory threat and a given rewarding stimulus, must make rapid decisions to engage in defensive versus other appetitive behaviors. The brain mechanisms mediating such responses are poorly understood. However, the periaqueductal gray (PAG) seems particularly suitable for accomplishing this task. The PAG is thought to have, at least, two distinct general roles on the organization of motivated responses, i.e., one on the execution of defensive and reproductive behaviors, and the other on the motivational drive underlying adaptive responses. We have presently examined how the PAG would be involved in mediating the behavioral choice between mutually incompatible behaviors, such as reproduction or defense, when dams are exposed to pups and cat odor. First, we established the behavioral protocol and observed that lactating rats, simultaneously exposed to pups and cat odor, inhibited maternal behavior and expressed clear defensive responses. We have further revealed that cat odor exposure up-regulated Fos expression in the dorsal PAG, and that NMDA cytotoxic lesions therein were able to restore maternal responses, and, at the same time, block defensive responsiveness to cat odor. Potential paths mediating the dorsal PAG influences on the inhibition of appetitive (i.e., retrieving behavior) and consummatory (i.e., nursing) maternal responses are discussed. Overall, we were able to confirm the dual role of the PAG, where, in the present case, the dorsal PAG, apart from organizing defensive responses, also appears to account for the behavioral inhibition of non-defensive responses. (C) 2010 Elsevier B.V. All rights reserved.

L-NAME Treatment Enhances Exercise-induced Content of Myocardial Heat Shock Protein 72 (Hsp72) in Rats

Background/Aim: Nitric oxide (NO) modulates the expression of the chaperone Hsp72 in the heart, and exercise stimulates both NO production and myocardial Hsp72 expression. The main purpose of the study was to investigate whether NO interferes with an exercise-induced myocardial Hsp72 expression. Methods: Male Wistar rats (70-100 days) were divided into control (C, n= 12), L-NAME-treated (L, n= 12), exercise (E, n= 13) and exercise plus L-NAME-treated (EL, n= 20) groups. L-NAME was given in drinking water (700 mg. L(-1)) and the exercise was performed on a treadmill (15-25 m.min(-1), 40-60 min. day(-1)) for seven days. Left ventricle (LV) protein Hsp content, NOS and phosphorylated-NOS (p-NOS) isoforms were measured using Western blotting. The activity of NOS was assayed in LV homogenates by the conversion of [(3)H] L-arginine to [(3)H] L-citrulline. Results: Hsp72 content was increased significantly (223%; p < 0.05) in the E group compared to the C group, but exercise alone did not alter the NOS content, p-NOS isoforms or NOS activity. Contrary to our expectation, L-NAME enhanced (p < 0.05) the exercise-induced Hsp72 content (EL vs. C, L and E groups = 1019%, 548% and 457%, respectively). Although the EL group had increased stimulatory p-eNOS(Ser1177) (over 200%) and decreased inhibitory p-nNOS(Ser852) (similar to 50%) compared to both the E and L groups (p < 0.05), NOS activity was similar in all groups. Conclusions: Our results suggest that exercise-induced cardiac Hsp72 expression does not depend on NO. Conversely, the in vivo L-NAME treatment enhances exercise-induced Hsp72 production. This effect may be due to an increase in cardiac stress. Copyright (C) 2011 S. Karger AG, Basel

Effect of low-level laser therapy (GaAs 904 nm) in skeletal muscle fatigue and biochemical markers of muscle damage in rats

We wanted to test if pre-exercise muscle irradiation with 904 nm laser affects the development of fatigue, blood lactate levels and creatine kinase (CK) activity in a rat model with tetanic contractions. Thirty male Wistar rats were divided into five groups receiving either one of four different laser doses (0.1, 0.3, 1.0 and 3.0 J) or a no-treatment control group. Laser irradiation was performed immediately before the first contraction for treated groups. Electrical stimulation was used to induce six tetanic tibial anterior muscle contractions with 10 min intervals between them. Contractions were stopped when the muscle force fell to 50% of the peak value for each contraction; blood samples were taken before the first and immediately after the sixth contraction. The relative peak forces for the sixth contraction were significantly better (P < 0.05) in the two laser groups irradiated with highest doses [151.27% (SD +/- A 18.82) for 1.0 J, 144.84% (SD +/- A 34.47) for 3.0 J and 82.25% (SD +/- A 11.69) for the control group]. Similar significant (P < 0.05) increases in mean performed work during the sixth contraction for the 1.0 and 3.0 J groups were also observed. Blood lactate levels were significantly lower (P < 0.05) than the control group in all irradiated groups. All irradiated groups except the 3.0 J group had significantly lower post-exercise CK activity than the control group. We conclude that pre-exercise irradiation with a laser dose of 1.0 J and 904 nm wavelength significantly delays muscle fatigue and decreases post-exercise blood lactate and CK in this rat model.

Effect of 830 nm low-level laser therapy applied before high-intensity exercises on skeletal muscle recovery in athletes

Our aim was to investigate the immediate effects of bilateral, 830 nm, low-level laser therapy (LLLT) on high-intensity exercise and biochemical markers of skeletal muscle recovery, in a randomised, double-blind, placebo-controlled, crossover trial set in a sports physiotherapy clinic. Twenty male athletes (nine professional volleyball players and eleven adolescent soccer players) participated. Active LLLT (830 nm wavelength, 100 mW, spot size 0.0028 cm(2), 3-4 J per point) or an identical placebo LLLT was delivered to five points in the rectus femoris muscle (bilaterally). The main outcome measures were the work performed in the Wingate test: 30 s of maximum cycling with a load of 7.5% of body weight, and the measurement of blood lactate (BL) and creatine kinase (CK) levels before and after exercise. There was no significant difference in the work performed during the Wingate test (P > 0.05) between subjects given active LLLT and those given placebo LLLT. For volleyball athletes, the change in CK levels from before to after the exercise test was significantly lower (P = 0.0133) for those given active LLLT (2.52 U l(-1) +/- 7.04 U l(-1)) than for those given placebo LLLT (28.49 U l(-1) +/- 22.62 U l(-1)). For the soccer athletes, the change in blood lactate levels from before exercise to 15 min after exercise was significantly lower (P < 0.01) in the group subjected to active LLLT (8.55 mmol l(-1) +/- 2.14 mmol l(-1)) than in the group subjected to placebo LLLT (10.52 mmol l(-1) +/- 1.82 mmol l(-1)). LLLT irradiation before the Wingate test seemed to inhibit an expected post-exercise increase in CK level and to accelerate post-exercise lactate removal without affecting test performance. These findings suggest that LLLT may be of benefit in accelerating post-exercise recovery.

Effect of Cluster Multi-Diode Light Emitting Diode Therapy (LEDT) on Exercise-Induced Skeletal Muscle Fatigue and Skeletal Muscle Recovery in Humans

Background and Objectives: There are some indications that low-level laser therapy (LLLT) may delay the development of skeletal muscle fatigue during high-intensity exercise. There have also been claims that LED cluster probes may be effective for this application however there are differences between LED and laser sources like spot size, spectral width, power output, etc. In this study we wanted to test if light emitting diode therapy (LEDT) can alter muscle performance, fatigue development and biochemical markers for skeletal muscle recovery in an experimental model of biceps humeri muscle contractions. Study Design/Materials and Methods: Ten male professional volleyball players (23.6 [SD +/- 5.6] years old) entered a randomized double-blinded placebo-controlled crossover trial. Active cluster LEDT (69 LEDs with wavelengths 660/850 nm, 10/30 mW, 30 seconds total irradiation time, 41.7J of total energy irradiated) or an identical placebo LEDT was delivered under double-blinded conditions to the middle of biceps humeri muscle immediately before exercise. All subjects performed voluntary biceps humeri contractions with a workload of 75% of their maximal voluntary contraction force (MVC) until exhaustion. Results: Active LEDT increased the number of biceps humeri contractions by 12.9% (38.60 [SD +/- 9.03] vs. 34.20 [SD +/- 8.68], P = 0.021) and extended the elapsed time to perform contractions by 11.6% (P = 0.036) versus placebo. In addition, post-exercise levels of biochemical markers decreased significantly with active LEDT: Blood Lactate (P = 0.042), Creatine Kinase (P = 0.035), and C-Reative Protein levels (P = 0.030), when compared to placebo LEDT. Conclusion: We conclude that this particular procedure and dose of LEDT immediately before exhaustive biceps humeri contractions, causes a slight delay in the development of skeletal muscle fatigue, decreases post-exercise blood lactate levels and inhibits the release of Creatine Kinase and C-Reative Protein. Lasers Surg. Med. 41:572-577, 2009. (C) 2009 Wiley-Liss, Inc.

Differential Antitumor Effects of IgG and IgM Monoclonal Antibodies and Their Synthetic Complementarity-Determining Regions Directed to New Targets of B16F10-Nex2 Melanoma Cells

Malignant melanoma has increased incidence worldwide and causes most skin cancer-related deaths. A few cell surface antigens that can be targets of antitumor immunotherapy have been characterized in melanoma. This is an expanding field because of the ineffectiveness of conventional cancer therapy for the metastatic form of melanoma. In the present work, antimelanoma monoclonal antibodies (mAbs) were raised against B16F10 cells (subclone Nex4, grown in murine serum), with novel specificities and antitumor effects in vitro and in vivo. MAb A4 (IgG2ak) recognizes a surface antigen on B16F10-Nex2 cells identified as protocadherin beta(13). It is cytotoxic in vitro and in vivo to B16F10-Nex2 cells as well as in vitro to human melanoma cell lines. MAb A4M (IgM) strongly reacted with nuclei of permeabilized murine tumor cells, recognizing histone 1. Although it is not cytotoxic in vitro, similarly with mAb A4, mAb A4M significantly reduced the number of lung nodules in mice challenged intravenously with B16F10-Nex2 cells. The V(H) CDR3 peptide from mAb A4 and V(L) CDR1 and CDR2 from mAb A4M showed significant cytotoxic activities in vitro, leading tumor cells to apoptosis. A cyclic peptide representing A4 CDR H3 competed with mAb A4 for binding to melanoma cells. MAb A4M CDRs L1 and L2 in addition to the antitumor effect also inhibited angiogenesis of human umbilical vein endothelial cells in vitro. As shown in the present work, mAbs A4 and A4M and selected CDR peptides are strong candidates to be developed as drugs for antitumor therapy for invasive melanoma.

Differential expression of cytokines, chemokines and chemokine receptors in patients with coronary artery disease

Monocytes/macrophages and lymphocytes have a key role in the pathogenesis of atherosclerosis through the production of inflammatory and anti-inflammatory cytokines. We evaluated mRNA expression and protein production of CCL2, CXCL8, CXCL9, CXCL10, IFN-gamma and IL-10 in vitro as well as the expression of the CCR2 and CXCR3 receptors in peripheral blood mononuclear cells (PBMCs) of patients with coronary artery disease (CAD) and healthy controls in the presence or absence of oxidized LDL (oxLDL). Patients with CAD showed higher constitutive expression of CCL2, CXCL8, CXCL9, CXCL10 and IFN-gamma mRNA and, after stimulation with oxLDL, higher expression of CCL2 and CXCL8 mRNA than the control group. We also detected higher levels of CCL2 and CXCL8 in supernatants of oxLDL-stimulated PBMCs from CAD patients than in corresponding supernatants from controls. Patients with CAD had a higher percentage of constitutive CCR2(+) and CXCR3(+) cells after stimulation with oxLDL. Among CAD patients, the main differences between the stable (SA) and unstable angina (UA) groups were lower IL-10 mRNA production in the latter group. Altogether, our data suggest that PBMCs from CAD patients are able to produce higher concentrations of chemokines and cytokines involved in the regulation of monocyte and lymphocyte migration and retention in atherosclerotic lesions. (C) 2008 Elsevier Ireland Ltd. All rights reserved.

Expression and purification of human respiratory syncytial virus recombinant fusion protein

The Human Respiratory Syncytial Virus (HRSV) fusion protein (F) was expressed in Escherichia call BL21A using the pET28a vector at 37 degrees C. The protein was purified from the soluble fraction using affinity resin. The structural quality of the recombinant fusion protein and the estimation of its secondary structure were obtained by circular dichroism. Structural models of the fusion protein presented 46% of the helices in agreement with the spectra by circular dichroism analysis. There are only few studies that succeeded in expressing the HRSV fusion protein in bacteria. This is a report on human fusion protein expression in E. call and structure analysis, representing a step forward in the development of fusion protein F inhibitors and the production of antibodies. (c) 2008 Elsevier Inc. All rights reserved.

Epidemiology and Genetic Variability of Human Metapneumovirus During a 4-Year-Long Study in Southeastern Brazil

Epidemiological and molecular characteristics of human metapneumovirus (hMPV) were compared with human respiratory syncytial virus (hRSV) in infants and young children admitted for acute lower respiratory tract infections in a prospective study during four consecutive years in subtropical Brazil. GeneScan polymerase chain assays (GeneScan RT-PCR) were used to detect hMPV and hRSV in nasopharyngeal aspirates of 1,670 children during January 2003 to December 2006. hMPV and hRSV were detected, respectively, in 191 (11.4%) and in 702 (42%) of the children admitted with acute lower respiratory tract infections at the Sao Paulo University Hospital. Sequencing data of the hMPV F gene revealed that two groups of the virus, each divided into two subgroups, co-circulated during three consecutive years. It was also shown that a clear dominance of genotype B1 occurred during the years 2004 and 2005, followed by genotype A2 during 2006. J. Med. Virol. 81:915-921,2009. (C) 2009 Wiley-Liss, Inc.

Prefrontal afferents to the dorsal raphe nucleus in the rat

The prefrontal cortex (PFC) receives strong inputs from monoaminergic cell groups in the brainstem and also sends projections to these nuclei. Recent evidence suggests that the PFC exerts a powerful top-down control over the dorsal raphe nucleus (DR) and that it may be involved in the actions of pharmaceutical drugs and drugs of abuse. In the light of these findings, the precise origin of prefrontal inputs to DR was presently investigated by using the cholera toxin subunit b (CTb) as retrograde tracer. All the injections placed in DR produced retrograde labeling in the medial, orbital, and lateral divisions of the PFC as well as in the medial part of the frontal polar cortex. The labeling was primarily located in layer V. Remarkably, labeling in the medial PFC was denser in its ventral part (infralimbic and ventral prelimbic cortices) than in its dorsal part (dorsal prelimbic, anterior cingulate and medial precentral cortices). After injections in the rostral or caudal DR, the largest number of labeled neurons was observed in the medial PFC, whereas after injections in the mid-rostrocaudal DR, the labeled neurons were more homogeneously distributed in the three main PFC divisions. A cluster of labeled neurons also was observed around the apex of the rostral pole of the accumbens, especially after rostral and mid-rostrocaudal DR injections. Overall, these results confirm the existence of robust preftontal projections to DR, mainly derived from the ventral part of the medial PFC, and underscore a substantial contribution of the frontal polar cortex. (C) 2008 Elsevier Inc. All rights reserved.