Astroglial cells derived from lateral and medial midbrain sectors differ in their synthesis and secretion of sulfated glycosaminoglycans

Astroglial cells derived from lateral and medial midbrain sectors differ in their abilities to support neuritic growth of midbrain neurons in cocultures. These different properties of the two types of cells may be related to the composition of their extracellular matrix. We have studied the synthesis and secretion of sulfated glycosaminoglycans (GAGs) by the two cell types under control conditions and ß-D-xyloside-stimulated conditions, that stimulate the ability to synthesize and release GAGs. We have confirmed that both cell types synthesize and secrete heparan sulfate and chondroitin sulfate. Only slight differences were observed between the proportions of the two GAGs produced by the two types of cells after a 24-h labeling period. However, a marked difference was observed between the GAGs produced by the astroglial cells derived from lateral and medial midbrain sectors. The medial cells, which contain derivatives of the tectal and tegmental midline radial glia, synthesized and secreted ~2.3 times more chondroitin sulfate than lateral cells. The synthesis of heparan sulfate was only slightly modified by the addition of ß-D-xyloside. Overall, these results indicate that astroglial cells derived from the two midbrain sectors have marked differences in their capacity to synthesize chondroitin sulfate. Under in vivo conditions or a long period of in vitro culture, they may produce extracellular matrix at concentrations which may differentially affect neuritic growth.

Metabolite and light regulation of metabolism in plants: lessons from the study of a single biochemical pathway

We are using molecular, biochemical, and genetic approaches to study the structural and regulatory genes controlling the assimilation of inorganic nitrogen into the amino acids glutamine, glutamate, aspartate and asparagine. These amino acids serve as the principal nitrogen-transport amino acids in most crop and higher plants including Arabidopsis thaliana. We have begun to investigate the regulatory mechanisms controlling nitrogen assimilation into these amino acids in plants using molecular and genetic approaches in Arabidopsis. The synthesis of the amide amino acids glutamine and asparagine is subject to tight regulation in response to environmental factors such as light and to metabolic factors such as sucrose and amino acids. For instance, light induces the expression of glutamine synthetase (GLN2) and represses expression of asparagine synthetase (ASN1) genes. This reciprocal regulation of GLN2 and ASN1 genes by light is reflected at the level of transcription and at the level of glutamine and asparagine biosynthesis. Moreover, we have shown that the regulation of these genes is also reciprocally controlled by both organic nitrogen and carbon metabolites. We have recently used a reverse genetic approach to study putative components of such metabolic sensing mechanisms in plants that may be conserved in evolution. These components include an Arabidopsis homolog for a glutamate receptor gene originally found in animal systems and a plant PII gene, which is a homolog of a component of the bacterial Ntr system. Based on our observations on the biology of both structural and regulatory genes of the nitrogen assimilatory pathway, we have developed a model for metabolic control of the genes involved in the nitrogen assimilatory pathway in plants.

Characterization of renin mRNA expression and enzyme activity in rat and mouse mesangial cells

Renin is an enzyme involved in the stepwise generation of angiotensin II. Juxtaglomerular cells are the main source of plasma renin, but renin activity has been detected in other cell types. In the present study we evaluated the presence of renin mRNA in adult male Wistar rat and mouse (C-57 Black/6) mesangial cells (MC) and their ability to process, store and release both the active and inactive forms of the enzyme. Active renin and total renin content obtained after trypsin treatment were estimated by angiotensinogen consumption analyzed by SDS-PAGE electrophoresis and quantified by angiotensin I generation by HPLC. Renin mRNA, detected by RT-PCR, was present in both rat and mouse MC under basal conditions. Active renin was significantly higher (P<0.05) in the cell lysate (43.5 ± 5.7 ng h-1 10(6) cells) than in the culture medium (12.5 ± 2.5 ng h-1 10(6) cells). Inactive prorenin content was similar for the intra- and extracellular compartments (9.7 ± 3.1 and 3.9 ± 0.9 ng h-1 10(6) cells). Free active renin was the predominant form found in both cell compartments. These results indicate that MC in culture are able to synthesize and translate renin mRNA probably as inactive prorenin which is mostly processed to active renin inside the cell. MC secrete both forms of the enzyme but at a lower level compared with intracellular content, suggesting that the main role of renin synthesized by MC may be the intracellular generation of angiotensin II.

Determination of anaerobic threshold in rats using the lactate minimum test

The break point of the curve of blood lactate vs exercise load has been called anaerobic threshold (AT) and is considered to be an important indicator of endurance exercise capacity in human subjects. There are few studies of AT determination in animals. We describe a protocol for AT determination by the "lactate minimum test" in rats during swimming exercise. The test is based on the premise that during an incremental exercise test, and after a bout of maximal exercise, blood lactate decreases to a minimum and then increases again. This minimum value indicates the intensity of the AT. Adult male (90 days) Wistar rats adapted to swimming for 2 weeks were used. The initial state of lactic acidosis was obtained by making the animals jump into the water and swim while carrying a load equivalent to 50% of body weight for 6 min (30-s exercise interrupted by a 30-s rest). After a 9-min rest, blood was collected and the incremental swimming test was started. The test consisted of swimming while supporting loads of 4.5, 5.0, 5.5, 6.0 and 7.0% of body weight. Each exercise load lasted 5 min and was followed by a 30-s rest during which blood samples were taken. The blood lactate minimum was determined from a zero-gradient tangent to a spline function fitting the blood lactate vs workload curve. AT was estimated to be 4.95 ± 0.10% of body weight while interpolated blood lactate was 7.17 ± 0.16 mmol/l. These results suggest the application of AT determination in animal studies concerning metabolism during exercise.

Cytoskeletal and cellular adhesion proteins in zebrafish (Danio rerio) myogenesis

The current myogenesis and myofibrillogenesis model has been based mostly on in vitro cell culture studies, and, to a lesser extent, on in situ studies in avian and mammalian embryos. While the more isolated artificial conditions of cells in culture permitted careful structural analysis, the actual in situ cellular structures have not been described in detail because the embryos are more difficult to section and manipulate. To overcome these difficulties, we used the optically clear and easy to handle embryos of the zebrafish Danio rerio. We monitored the expression of cytoskeletal and cell-adhesion proteins (actin, myosin, desmin, alpha-actinin, troponin, titin, vimentin and vinculin) using immunofluorescence microscopy and video-enhanced, background-subtracted, differential interference contrast of 24- to 48-h zebrafish embryos. In the mature myotome, the mononucleated myoblasts displayed periodic striations for all sarcomeric proteins tested. The changes in desmin distribution from aggregates to perinuclear and striated forms, although following the same sequence, occurred much faster than in other models. All desmin-positive cells were also positive for myofibrillar proteins and striated, in contrast to that which occurs in cell cultures. Vimentin appeared to be striated in mature cells, while it is developmentally down-regulated in vitro. The whole connective tissue septum between the somites was positive for adhesion proteins such as vinculin, instead of the isolated adhesion plaques observed in cell cultures. The differences in the myogenesis of zebrafish in situ and in cell culture in vitro suggest that some of the previously observed structures and protein distributions in cultures could be methodological artifacts.

Validation of a simple and inexpensive method for the quantitation of infarct in the rat brain

A gravimetric method was evaluated as a simple, sensitive, reproducible, low-cost alternative to quantify the extent of brain infarct after occlusion of the medial cerebral artery in rats. In ether-anesthetized rats, the left medial cerebral artery was occluded for 1, 1.5 or 2 h by inserting a 4-0 nylon monofilament suture into the internal carotid artery. Twenty-four hours later, the brains were processed for histochemical triphenyltetrazolium chloride (TTC) staining and quantitation of the schemic infarct. In each TTC-stained brain section, the ischemic tissue was dissected with a scalpel and fixed in 10% formalin at 0ºC until its total mass could be estimated. The mass (mg) of the ischemic tissue was weighed on an analytical balance and compared to its volume (mm³), estimated either by plethysmometry using platinum electrodes or by computer-assisted image analysis. Infarct size as measured by the weighing method (mg), and reported as a percent (%) of the affected (left) hemisphere, correlated closely with volume (mm³, also reported as %) estimated by computerized image analysis (r = 0.88; P < 0.001; N = 10) or by plethysmography (r = 0.97-0.98; P < 0.0001; N = 41). This degree of correlation was maintained between different experimenters. The method was also sensitive for detecting the effect of different ischemia durations on infarct size (P < 0.005; N = 23), and the effect of drug treatments in reducing the extent of brain damage (P < 0.005; N = 24). The data suggest that, in addition to being simple and low cost, the weighing method is a reliable alternative for quantifying brain infarct in animal models of stroke.

Becoming a caring nurse : the heart of the matter in nurse education

This thesis in caring science didactics is based on a thinking, where the fundamental basis for the didactic is science-based, i.e. it does not emanate from the nursing profession but brings forward a didactic that grows out of caring science and its core substance and ethos. This view on didactics arises from the caritative theory developed by Eriksson. The overall aim of the study is to clarify the meaning and essence of understanding, as well as to explore and deepen the understanding of student nurses' processes of understanding and becoming with the intention of developing a theory model for caring didactics. The overarching research questions are: What is the essence of understanding (of caring science knowledge)? What are the possibilities and importance of understanding in the appropriation of caring science? What characterizes and impels the process of becoming? The thesis consists of four sub studies and a summary section. The overall methodological approach is hermeneutic involving quantitative as well as qualitative methods. The data for the study has been collected through a longitudinal research project that followed student nurses at three universities during their entire education. The empirical sub studies form the basis for the interpreted knowledge that is formulated in the new understanding. This new understanding have, through additional theory-charging with the theory fragments from Gadamer, generated the heuristic synthesis which is illustrated in the theory model. The findings shows that understanding can be described as something unlimited, as an endless movement, which can be illustrated as a lying eighth, a lemniscate. The lemiscate of understanding is characterized by seeing, knowing and becoming and consists of seven differently named phases; the acquired horizon of understanding, the encounter of horizons, the dialogue of horizons, the fusion of horizons, application, reflection and shaping a new horizon of understanding. Bildung (formation), is the ultimate imprint of the endless spiral movement of understanding. Ethos and arête constitute the hubs around which the lemniscate of understanding circles. These include the spirit and driving force that the student carries within. The caring culture encloses the lemniscate of understanding. The caring culture provides the life space of understanding and the prevailing basic values are evinced in the culture.

Respiration, oxidative phosphorylation, and uncoupling protein in Candida albicans

The respiration, membrane potential (Dy), and oxidative phosphorylation of mitochondria in situ were determined in spheroplasts obtained from Candida albicans control strain ATCC 90028 by lyticase treatment. Mitochondria in situ were able to phosphorylate externally added ADP (200 µM) in the presence of 0.05% BSA. Mitochondria in situ generated and sustained stable mitochondrial Dy respiring on 5 mM NAD-linked substrates, 5 mM succinate, or 100 µM N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride plus 1 mM ascorbate. Rotenone (4 µM) inhibited respiration by 30% and 2 µM antimycin A or myxothiazole and 1 mM cyanide inhibited it by 85%. Cyanide-insensitive respiration was partially blocked by 2 mM benzohydroxamic acid, suggesting the presence of an alternative oxidase. Candida albicans mitochondria in situ presented a carboxyatractyloside-insensitive increase of Dy induced by 5 mM ATP and 0.5% BSA, and Dy decrease induced by 10 µM linoleic acid, both suggesting the existence of an uncoupling protein. The presence of this protein was subsequently confirmed by immunodetection and respiration experiments with isolated mitochondria. In conclusion, Candida albicans ATCC 90028 possesses an alternative electron transfer chain and alternative oxidase, both absent in animal cells. These pathways can be exceptional targets for the design of new chemotherapeutic agents. Blockage of these respiratory pathways together with inhibition of the uncoupling protein (another potential target for drug design) could lead to increased production of reactive oxygen species, dysfunction of Candida mitochondria, and possibly to oxidative cell death.

Effect of Sapindus trifoliatus on hyperalgesic in vivo migraine models

Phytotherapies have offered alternative sources of therapy for migraine and gained much importance in prophylactic treatment. Sapindus trifoliatus is a medium-sized deciduous tree growing wild in south India that belongs to the family Sapindaceae. The pericarp is reported for various medicinal properties. A thick aqueous solution of the pericarp is used for the treatment of hemicrania, hysteria or epilepsy in folklore medicine. We have investigated the antihyperalgesic effects of the lyophilized aqueous extract of S. trifoliatus in animal models predictive of experimental migraine models using morphine withdrawal-induced hyperalgesia on the hot-plate test and on 0.3% acetic acid-induced abdominal constrictions in adult male Swiss albino mice. The extract significantly (N = 10, P < 0.05) increased the licking latency in the hot-plate test when administered ip at 10 mg/kg (6.70 ± 0.39 s in saline control vs 18.76 ± 0.96 s in S. trifoliatus-treated animals) and significantly (N = 10, P < 0.001) reduced the abdominal constrictions when administered ip at 2 and 10 mg/kg (40.20 ± 1.36 in saline control vs 30.20 ± 1.33 and 23.00 ± 0.98 for 2 and 10 mg/kg, ip, respectively, in S. trifoliatus-treated animals). Furthermore, when administered ip at 20 and 100 mg/kg, the extract significantly (N = 10, P < 0.05) inhibited the apomorphine-induced climbing behavior in mice (climbing duration 15.75 ± 5.0 min for saline control vs 11.4 ± 1.28 and 3.9 ± 1.71 min for 20 and 100 mg/kg, respectively, in S. trifoliatus-treated animals). In receptor radioligand-binding studies, the extract exhibited affinity towards D2 receptors. The findings suggest that dopamine D2 antagonism could be the mechanism involved in the antihyperalgesic activity of the aqueous extract of S. trifoliatus.

Maturation of pig oocytes in vitro in a medium with pyruvate

The aim of in vitro maturation oocyte systems is to produce oocytes of comparable quality to those derived in vivo. The present study was designed to examine the surface morphological changes of the cumulus-oocyte complex (COC) and nuclear maturation in a culture system containing pyruvate. Ovaries were obtained from a slaughterhouseand transported to the laboratory within 2 h at 35-39ºC,and rinsed three times in 0.9% NaCl. The COCs were harvested from the ovaries and in vitro maturation was evaluated in San Marcos (SM) medium, a chemically defined culture system containing 22.3 mM sodium pyruvate. Oocytes were cultured in SM, SM + porcine follicular fluid (pFF) and in SM + pFF + gonadotropins (eCG and hCG) for 20-22 h and then without hormonal supplements for an additional 20-22 h. After culture, the degree of cumulus expansion and frequency of nuclear maturation were determined. Oocytes matured in SM (40.9%) and SM + pFF (42.9%) showed moderate cumulus expansion, whereas oocytes matured in SM + pFF + gonadotropins (54.6%) showed high cumulus expansion. The maturation rate of cultured oocytes, measured in function of the presence of the polar corpuscle, did not differ significantly between SM (40.9 ± 3.6%) and SM + pFF (42.9 ± 3.7%). These results indicate that pig oocytes can be successfully matured in a chemically definedmedium and suggest a possible bifunctional role of pyruvate as an energy substrate and as an antioxidant protecting oocytes against the stress of the in vitro environment.

Inactivation of Staphylococcus aureus and Salmonella enteritidis in tryptic soy broth and caviar samples by high pressure processing

We studied the action of high pressure processing on the inactivation of two foodborne pathogens, Staphylococcus aureus ATCC 6538 and Salmonella enteritidis ATCC 13076, suspended in a culture medium and inoculated into caviar samples. The baroresistance of the two pathogens in a tryptic soy broth suspension at a concentration of 10(8)-10(9) colony-forming units/ml was tested for continuous and cycled pressurization in the 150- to 550-MPa range and for 15-min treatments at room temperature. The increase of cycle number permitted the reduction of the pressure level able to totally inactivate both microorganisms in the tryptic soy broth suspension, whereas the effect of different procedure times on complete inactivation of the microorganisms inoculated into caviar was similar.

Neo-Timm staining in the thalamus of chronically epileptic rats

The thalamus is an important modulator of seizures and is severely affected in cholinergic models of epilepsy. In the present study, chronically epileptic rats had their brains processed for neo-Timm and acetylcholinesterase two months after the induction of status epilepticus with pilocarpine. Both controls and pilocarpine-treated animals presented neo-Timm staining in the anterodorsal nucleus, laterodorsal nucleus, reticular nucleus, most intralaminar nuclei, nucleus reuniens, and rhomboid nucleus of the thalamus, as well as in the zona incerta. The intensity of neo-Timm staining was similar in control and pilocarpine-treated rats, except for the nucleus reuniens and the rhomboid nucleus, which had a lower intensity of staining in the epileptic group. In animal models of temporal lobe epilepsy, zinc seems to modulate glutamate release and to decrease seizure activity. In this context, a reduction of neo-Timm-stained terminals in the midline thalamus could ultimately result in an increased excitatory activity, not only within its related nuclei, but also in anatomical structures that receive their efferent connections. This might contribute to the pathological substrate observed in chronic pilocarpine-treated epileptic animals.

The RNA interference revolution

The discovery of double-stranded RNA-mediated gene silencing has rapidly led to its use as a method of choice for blocking a gene, and has turned it into one of the most discussed topics in cell biology. Although still in its infancy, the field of RNA interference has already produced a vast array of results, mainly in Caenorhabditis elegans, but recently also in mammalian systems. Micro-RNAs are short hairpins of RNA capable of blocking translation, which are transcribed from genomic DNA and are implicated in several aspects from development to cell signaling. The present review discusses the main methods used for gene silencing in cell culture and animal models, including the selection of target sequences, delivery methods and strategies for a successful silencing. Expected developments are briefly discussed, ranging from reverse genetics to therapeutics. Thus, the development of the new paradigm of RNA-mediated gene silencing has produced two important advances: knowledge of a basic cellular mechanism present in the majority of eukaryotic cells and access to a potent and specific new method for gene silencing.

Immunoglobulin production is impaired in protein-deprived mice and can be restored by dietary protein supplementation

Most contacts with food protein and microbiota antigens occur at the level of the gut mucosa. In animal models where this natural stimulation is absent, such as germ-free and antigen-free mice, the gut-associated lymphoid tissue (GALT) and systemic immunological activities are underdeveloped. We have shown that food proteins play a critical role in the full development of the immune system. C57BL/6 mice weaned to a diet in which intact proteins are replaced by equivalent amounts of amino acids (Aa diet) have a poorly developed GALT as well as low levels of serum immunoglobulins (total Ig, IgG, and IgA, but not IgM). In the present study, we evaluated whether the introduction of a protein-containing diet in 10 adult Aa-fed C57BL/6 mice could restore their immunoglobulin levels and whether this recovery was dependent on the amount of dietary protein. After the introduction of a casein-containing diet, Aa-fed mice presented a fast recovery (after 7 days) of secretory IgA (from 0.33 to 0.75 mg/mL, while in casein-fed mice this value was 0.81 mg/mL) and serum immunoglobulin levels (from 5.39 to 10.25 mg/mL of total Ig). Five percent dietary casein was enough to promote the restoration of secretory IgA and serum immunoglobulin levels to a normal range after 30 days feeding casein diet (as in casein-fed mice - 15% by weight of diet). These data suggest that the defect detected in the immunoglobulin levels was a reversible result of the absence of food proteins as an antigenic stimulus. They also indicate that the deleterious consequences of malnutrition at an early age for some immune functions may be restored by therapeutic intervention later in life.

Sex differences in angiotensin II- induced hypertension

Sex differences in the development of hypertension and cardiovascular disease have been described in humans and in animal models. In this paper we will review some of our studies which have as their emphasis the examination of the role of sex differences and sex steroids in modulating the central actions of angiotensin II (ANG II) via interactions with free radicals and nitric oxide, generating pathways within brain circumventricular organs and in central sympathomodulatory systems. Our studies indicate that low-dose infusions of ANG II result in hypertension in wild-type male mice but not in intact wild-type females. Furthermore, we have demonstrated that ANG II-induced hypertension in males is blocked by central infusions of the androgen receptor antagonist, flutamide, and by central infusions of the superoxide dismutase mimetic, tempol. We have also found that, in comparison to females, males show greater levels of intracellular reactive oxygen species in circumventricular organ neurons following long-term ANG II infusions. In female mice, ovariectomy, central blockade of estrogen receptors or total knockout of estrogen a receptors augments the pressor effects of ANG II. Finally, in females but not in males, central blockade of nitric oxide synthase increases the pressor effects of ANG II. Taken together, these results suggest that sex differences and estrogen and testosterone play important roles in the development of ANG II-induced hypertension.