Organization of the Subdiaphragmatic Vagus Nerve and Its Connection with the Celiac Plexus and the Ovaries in the Female Rat.

Communication between the ovaries and the central nervous system occurs by peripheral innervation through the celiac plexus, superior ovarian nerve, and ovarian plexus nerve. The vagus nerve is involved in regulating the ovaries, but the neuroanatomical pathway that links them is not clear. Adult female rats were used for gross anatomy, acetylcholinesterase histochemistry, and the immunofluorescence analysis of tyrosine hydroxylase (TH), choline acetyltransferase (ChAT), and tryptophan hydroxylase 2 (TPH). The results obtained indicate that the right vagus nerve (RVN) travels parallel and caudal to the esophagus, where three nerve branches were identified. Also, a right vagal plexus (RVP) formed by microganglia was described, establishing communication with the celiac plexus, and was mainly reactive to tyrosine hydroxylase (TH); some serotoninergic and cholinergic neurons were also found. The left vagus nerve (LVN) travels over the esophagus, bifurcates before its insertion into the stomach and enters the RCG. This neuroanatomical and biochemical description of the RVN and LVN in the rat suggests the RVP is formed by presynaptic catecholaminergic terminals and cholinergic neurons. This information could support detailed studies of communication between the vagus nerve and the ovaries and identify the type of neural signaling involved in abdominal control of the vagus nerve.

Structural organization of the neuronal pathways of the superior ovarian nerve in the rat.

BACKGROUND: In the rat, studies have shown that ovary innervation arrives via the superior ovarian nerve (SON) and the ovarian plexus nerve, which originates from the celiac plexus (CP). In the present study, we performed a neuroanatomical technique to investigate the anatomy of the SON between the ovary and the CP. RESULTS: We found that the SON fibers were concentrated on the lateral border of the suprarenal ganglion and projected towards, then inserted into the suspensory ligament. Then, it ran parallel to the long axis of the ligament to reach and innervate the ovaries. At this level, the SON was composed of two coiled nerve fibers, each between 10 and 15 µm in diameter. The SON was linked to three different ganglia: the suprarenal ganglia, the celiac ganglia, and the superior mesenteric ganglion. CONCLUSIONS: The postganglionic fibers that project to the ovary via the SON emerge from the suprarenal ganglia. The trajectories on the right and left sides to each ovary are similar. The somas of ipsilateral and contralateral SON neurons are located in the prevertebral ganglia, mostly in the celiac ganglia.

Denatonium and Naringenin Promote SCA-9 Tumor Growth and Angiogenesis: Participation of Arginase.

Submandibular gland (SMG) is one of the major salivary glands, and is formed by acinar cells that are conveyed to the oral cavity by a duct system. We had previously reported that T2R receptors that were originally identified in gustatory tissues were also present in murine SMG. The addition of bitter compounds to the gland reduced nitric oxide production and downregulated amylase secretion. In this work, we investigated the effect of two different bitter compounds namely denatonium and naringenin on tumor progression as well as the presence of T2R in SCA-9 cells derived from a murine tumor induced in SMG. Both compounds increased tumor cell proliferation in bi- and three-dimensional cultures. These effects were mediated by the activation of arginase and the inhibition of nitric oxide synthase. Denatonium and naringenin also increased vascular endothelial growth factor-A expression via arginase and tumor neovascularization in vivo. T2R6 and T2R4 were identified in SCA-9 cells by immunostaining. Also, Gi and Ggust proteins, which usually couple to T2R receptors, are expressed in these cells. Finally, we demonstrated for the first time that bitter compounds can exert pro-tumor actions that should be taken into account as side effects when they are used as nutraceuticals.

Macrophages derived from septic mice modulate nitric oxide synthase and angiogenic mediators in the heart.

Macrophages (Mps) can exert the defense against invading pathogens. During sepsis, bacterial lipopolisaccharide (LPS) activates the production of inflammatory mediators by Mps. Nitric oxide synthase (NOS) derived-nitric oxide (NO) is one of them. Besides, Mps may produce pro-angiogenic molecules such as vascular endothelial growth factor-A (VEGF-A) and metalloproteinases (MMPs). The mechanisms involved in the cardiac neovascular response by Mps during sepsis are not completely known. We investigated the ability of LPS-treated Mps from septic mice to modulate the behavior of cardiac cells as producers of NO and angiogenic molecules. In vivo LPS treatment (0.1 mg/mouse) increased NO production more than fourfold and induced de novo NOS2 expression in Mps. Immunoblotting assays also showed an induction in VEGF-A and MMP-9 expression in lysates obtained from LPS-treated Mps, and MMP-9 activity was detected by zymography in cell supernatants. LPS-activated Mps co-cultured with normal heart induced the expression of CD31 and VEGF-A in heart homogenates and increased MMP-9 activity in the supernatants. By immunohistochemistry, we detected new blood vessel formation in hearts cultured with LPS treated Mps. When LPS-stimulated Mps were co-cultured with isolated cardiomyocytes in a transwell assay, the expression of NOS2, VEGF-A and MMP-9 was induced in cardiac cells. In addition, MMP-9 activity was up-regulated in the supernatant of cardiomyocytes. The latter was due to NOS2 induction in Mps from in vivo LPS-treated mice. In conclusion LPS-treated Mps are inducers of inflammatory/angiogenic mediators in cardiac cells, which could be triggering neovascularization, as an attempt to improve cardiac performance in sepsis.

Loss-of-function genetic screening identifies a cluster of ribosomal proteins regulating p53 function.

Introduction of conditional murine p53 (p53val135) and oncogenic ras into double p53/p21-null mouse embryonic fibroblasts (MEFs) showed that p21waf1 was not required for combined ras/p53-induced senescent-like growth arrest. We used this cellular system to identify key players in the ras-p53-induced senescence in the absence of p21. Applying a retroviral-based genetic screen, we obtained mRNA antisense fragments against a cluster of 14 different ribosomal proteins which loss of function bypasses p53-induced growth arrest. The expression of the ribosomal protein antisense fragments reduced the transcriptional activity of p53. Experiments with eGFP-p53 chimeras suggest that the effect is mediated by a reduction of p53. To study whether p53 was downregulated by MDM2-dependent degradation, we tested the effect of the RP antisenses in double p53/MDM2-null MEFs and observed that in the absence of MDM2, reduction of the RP levels also decreases p53 levels. Therefore, although we cannot discard other unknown mechanism, we suggest that the decrease in the levels of ribosomal proteins might inhibit p53-specific translation. Finally, quantitative analysis comparing levels of mRNA in tumours versus mRNA in normal tissue of the same organ and patient showed that a variable percentage of lung, prostate or colon tumours have reduced levels of the RPs tested. Interestingly, in most cases, the reduction of ribosomal protein mRNAs occurs only to 50%. Our data suggest that ribosomal protein imbalance might contribute to p53 regulation through the ribosomal biogenesis checkpoint.

PPP1CA contributes to the senescence program induced by oncogenic Ras.

Ectopic expression of conditional murine p53 (p53val135) and oncogenic ras is enough to induce a senescent-like growth arrest at the restrictive temperature. We took advantage of this cellular system to identify new key players in the ras/p53-induced senescence. Applying a retroviral-based genetic screen, we obtained an antisense RNA fragment against PPP1CA, the catalytic subunit of protein phosphatase 1alpha, whose loss of function bypasses ras/p53-induced growth arrest and senescence. Expression of a specific short hairpin (sh)RNA against PPP1CA impairs the p53-dependent induction of p21 after DNA damage and blocks the subsequent pRb dephosphorylation, thus bypassing p53-induced arrest. We found that oncogenic ras promotes an increase in the intracellular level of ceramides together with an increase in the PPP1CA protein levels. Addition of soluble ceramide to the cells induced a senescence phenotype that is blocked through PPP1CA downregulation by specific shRNA. Analysis of human tumors suggests that one of the PPP1CA alleles might be lost in a high percentage of carcinomas such as kidney and colorectal. The overexpression of two out of five PPP1CA alternative spliced variants reduced tumor cell growth and the downregulation of the protein to hemizygosity increased the anchorage-independent growth. We propose that oncogenic stress induced by ras causes ceramide accumulation, therefore, increasing PPP1CA activity, pRb dephosphorylation and onset of the p53-induced arrest, contributing to tumor suppression.

Stimulus specificity in the acquisition and extinction of conditioned taste aversion.

An experiment evaluated whether the acquisition and extinction of conditioned taste aversion in the rat is stimulus-specific by testing the degree of response transfer between sweet and salty tastes. Animals in the paired-same and paired-different groups received a presentation of a gustatory CS and a cyclophosphamide injection US. Nonconditioned control groups received unpaired CS /US presentations or the CS followed by a vehicle injection. Taste avoidance was evaluated in three nonreinforced test sessions. In the paired-same, unpaired and vehicle groups, all test sessions were conducted with the same flavor as originally used in training, whereas the paired-different group was tested with a novel flavor on the first and second sessions and with the originally trained flavor in last session. Stimulus specific acquisition was apparent in the first test session, when the animals in the group paired-same exhibited lower fluid intake than the other three groups. Evidence of specificity of extinction was apparent in the last test session, when animals in the group paired-different exhibited lower fluid intake than the other three groups. These results provide further evidence of stimulus specificity in acquisition and extinction of conditioned taste aversion, supporting the associative interpretation of these phenomena.

Enhancement of Pavlovian conditioned immunosuppression in rats.

The goal of this study was to define conditions under which conditioned immunosuppression may be observed reliably. In three experiments, rats were exposed to a gustatory conditioned stimulus (CS) paired with cyclophosphamide (US), which induces immunosuppression and malaise. In Experiment 1, a single pairing of the CS with low, medium, or high doses of cyclophosphamide in separate groups produced no reliable conditioned immunosuppression even though conditioned taste aversion was observed in groups trained with high and medium doses of CY. Experiment 2 replicated the lack of effect following a single pairing of the CS with the medium dose of cyclophosphamide but demonstrated that three pairings are sufficient to induce conditioned immunosuppression. Experiment 3 demonstrated that significant immunosuppression is observable following a single CS-US pairing if the CS is presented in compound with a previously nonreinforced CS during training, an effect reminiscent of supernormal conditioning. These findings indicate that conditioned immunosuppression effects can be enhanced in magnitude through the use of certain procedural techniques.

Stimulus specificity in the acquisition and extinction of conditioned taste aversion

An experiment evaluated whether the acquisition and extinction of conditioned taste aversion in the rat is stimulus-specific by testing the degree of response transfer between sweet and salty tastes. Animals in the paired-same and paired-different groups received a presentation of a gustatory CS and a cyclophosphamide injection US. Nonconditioned control groups received unpaired CS /US presentations or the CS followed by a vehicle injection. Taste avoidance was evaluated in three nonreinforced test sessions. In the paired-same, unpaired and vehicle groups, all test sessions were conducted with the same flavor as originally used in training, whereas the paired-different group was tested with a novel flavor on the first and second sessions and with the originally trained flavor in last session. Stimulus specific acquisition was apparent in the first test session, when the animals in the group paired-same exhibited lower fluid intake than the other three groups. Evidence of specificity of extinction was apparent in the last test session, when animals in the group paired-different exhibited lower fluid intake than the other three groups. These results provide further evidence of stimulus specificity in acquisition and extinction of conditioned taste aversion, supporting the associative interpretation of these phenomena.

Cell Culture Derived AgMNPV Bioinsecticide: Biological Constraints and Bioprocess Issues.

We have studied parameters for optimizing the Spodoptera frugiperda (Sf9) cell culture and viral infection for the production of Anticarsia gemmatalis multiple nucleopolyhedrosis virus (AgMNPV) polyhedra inclusion bodies (PIBs) in shaker-Schott or spinner bottles and bioreactors. We have assayed the k(L)a of the systems, initial cell seeding, cell culture volume, dissolved oxygen (DO), multiplicity of infection (MOI), nutrients consumption, and metabolites production. The medium surface oxygen transfer was shown to be higher in shaker bottles than in spinner ones, which was in direct correlation to the higher cell density obtained. Best quantitative performances of PIBs production were obtained with a SF900II medium volume/shaker-bottle volume ratio of 15% and MOI of 0.5 to 1 performed at a cell concentration at infection (CCI) of 1 to 2.5x10(6) cells/ml in a medium containing enough glucose and glutamine. Upon infection, a decrease in the cell multiplication was observed to be dependent on the MOI used, and the muX at the exponential growth phase in infected and non-infected cultures were, respectively, of 0.2832 and 0.3914 (day(-1)). The glucose consumption and lactate production were higher in the infected cultures (muGlucose and muLactate of, respectively, 0.0248 and 0.0089x10(-8) g/cellxday in infected cultures and 0.0151 and 0.0046x10(-8) g/cellxday in non infected ones). The glutamine consumption did not differ in both cultures (muGlutamine of 0.0034 and 0.0037x10(-8) g/cellxday in, respectively, infected and non infected cultures). When a virus MOI of 0.1 to 1 was used for infection, a higher concentration of PIBs/ml was obtained. This was in direct correlation to a higher cell concentration present in these cultures, where a decrease in cell multiplication due to virus infection is minimized. When a MOI of 1 was used, a more effective decrease in cell multiplication was observed and a lower concentration of PIBs/ml was obtained, but with the best performance of PIBs/cell. Correlations between MOI and CCI indicate that a MOI 0.1 to 1.4 and a CCI of 10(6) to 2x10(6) cells/ml led to the best PIBs production performances. The virulence of PIBs produced in cultures infected at low or high MOI showed comparable DL(50). Culture and infection in scaling-up conditions, performed in a bioreactor, were shown to provide the cells with a better environment and be capable of potentially improving the shaker-Schott findings. For an accurate qualitative control of PIB virulence, hemolymph from AgMNPV infected Anticarsia gemmatalis was used as starting material for passages in Sf9 cells. These led to a loss of virulence among the PIBs with an increase in the DL(50). The loss of virulence was accompanied by a loss in budded virus titer, a decreased number of PIBs produced and an altered DNA restriction pattern, suggesting the generation of defective interference particles (DIPs). Transmission electron microscopy (TEM) studies revealed that after cell passages, PIBs lacking virions were progressively synthesized. The study described here point out the biological constraints and bioprocess issues for the preparation of AgMNPV PIBs for biological control.

Cellular senescence induced by p53-ras cooperation is independent of p21waf1 in murine embryo fibroblasts.

Oncogenic activation in primary murine fibroblasts initiates a senescence-like cell cycle arrest that depends on the p53 tumor suppressor pathway. Conditional p53 activation efficiently induced a reversible cell cycle arrest but was unable to induce features of senescence. In contrast, coexpression of oncogenic ras with p53 produced an irreversible cell cycle arrest that displayed features of cellular senescence. Introduction of a conditional murine p53 allele (p53val135) into double p53/p21-null mouse embryonic fibroblasts showed that p21waf1 was not required for this effect, since p53-/-;p21-/- double-null cells undergo terminal growth arrest with features of senescence following coexpression of oncogenic Ras and p53. Our results indicate that oncogenic activation of the Ras pathway in murine fibroblasts converts p53 into a senescence inducer through a p21waf1-independent mechanism.

Quantitative models of Pavlovian conditioning.

Over the last few years, research on learning and memory has become increasingly interdisciplinary. In the past, theories of learning, as a prerogative of psychologists, were generally formulated in purely verbal terms and evaluated exclusively at the behavioral level. At present, scientists are trying to build theories with a quantitative and biological flavor, seeking to embrace more complex behavioral phenomena. Pavlovian conditioning, one of the simplest and ubiquitous forms of learning, is especially suited for this multiple level analysis (i.e., quantitative, neurobiological, and behavioral), in part because of recent discoveries showing a correspondence between behavioral phenomena and associative properties at the cellular and systems levels, and in part because of its well established quantitative theoretical tradition. The present review, examines the mayor quantitative theories of Pavlovian conditioning and the phenomena to which they have been designed to account. In order to provide researchers from different disciplines with a simple guideline about the rationale of the different theoretical choices, all the models are described through a single formalism based on the neural network connectionist perspective.

Outbreak of carbapenem-resistant Acinetobacter baumannii producing the OXA-23 enzyme in Curitiba, Brazil.

Carbapenem-resistant Acinetobacter baumannii isolates were obtained from eight patients in two hospitals in Curitiba, Brazil. The isolates were multiresistant, belonged to a single strain, and produced the OXA-23 carbapenemase. Treatment options were limited, although the isolates were susceptible to polymyxin B in vitro. The strain contributed to the deaths of five patients.