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PubMed1990

Cells of origin of the spinohypothalamic tract in the rat.

We recently demonstrated that large numbers of neurons in the spinal cord of rats project directly to the hypothalamus. In the present study, we used the retrograde tracer Fluoro-Gold (FG) to examine this projection more completely. In the first series of studies, we attempted to label the entire population of spinal cord neurons that project to the hypothalamus. Injections that virtually filled the hypothalamus on one side without spreading into any other diencephalic area labeled a large number of neurons (estimated to be more than 9,000 in the case with the most neurons labeled) bilaterally at all levels of the spinal cord. Approximately 60% of the labeled neurons were contralateral to the injection. The greatest number of labeled neurons was found within the deep dorsal horn. Many were also found within the lateral spinal nucleus, the superficial dorsal horn, and the gray matter surrounding the central canal. A small number of labeled cells was located in the intermediate zone and ventral horn of the spinal gray matter. Labeled neurons were distributed bilaterally within the sacral parasympathetic nucleus and trigeminal nucleus caudalis. Injections of FG restricted to the medial hypothalamus labeled neurons within the spinal cord in a distribution similar to that produced by injections that filled the hypothalamus. However, fewer neurons were labeled in the spinal cord (estimated to be more than 6,200) and trigeminal nucleus caudalis. Injections of FG restricted to the lateral hypothalamus also labeled fewer neurons (approximately 3,300) than did injections that filled the hypothalamus. In these cases, also, the pattern of labeled neurons within the spinal cord was similar to that produced by injections within either medial or both medial and lateral hypothalamus. However, few neurons were labeled in the sacral parasympathetic nucleus following injections into the lateral hypothalamus. These findings show the distribution of a large number of spinal cord neurons that project directly to medial or lateral hypothalamic regions that are involved in autonomic, neuroendocrine, and emotional responses to somatosensory stimulation, including painful stimuli.

Afferent Pathways↗

PubMed1990

Morphological and physiological characterization of descending ocellar interneurons in the American cockroach.

In the present study two types of descending ocellar neurons have been morphologically and physiologically identified in the cockroach Periplaneta americana: a descending ipsilateral ocellar neuron (DIO-neuron) and a descending contralateral ocellar neuron (DCO-neuron). Both DIO- and DCO-neurons possess a cell body near the ocellar tract in the protocerebrum and extend dendritic processes into the ipsilateral ocellar nerve. An axon of the DIO-neuron descends to the metathoracic ganglion, and its entire course is ipsilateral to the cell body. An axon of the DCO-neuron crosses the median plane of the protocerebrum and descends through the contralateral ventral nerve cord to the second abdominal ganglion. Both axons possess masses of branches in each ganglion as well as in the brain. Both DIO- and DCO-neurons showed no spontaneous spike discharges, and responded with a few off-spikes to ocellar illumination. They responded to various mechanical stimuli, like cercal stimulation with a train of spikes which was suppressed by ocellar illumination. Spikes of DCO-neurons caused postsynaptic potentials and spikes in some interganglionic metathoracic interneurons, and spikes in motor nerves. These data are discussed in relation to the ocellar function of the cockroach as well as in comparison with that of other insects.

Animals↗

PubMed1990

Continuous cell activation is necessary for stable interaction of complement receptor type 3 with its counter-structure in the aggregation response of human neutrophils.

Human neutrophils aggregate after stimulation with various stimuli; this response is completely absent in neutrophils from patients with leukocyte adhesion deficiency (LAD). To investigate the cellular requirements of this process a method was used in which neutrophils are separately loaded with hydroethidine (HE) and sulfofluorescein (SFDA), to give them either red fluorescence or green fluorescence. After mixing HE- and SFDA-labeled cells in a ratio of 1:1, the number of double-colored aggregates formed after activation was determined by analysis on a fluorescence-activated cell sorter. In this way, essential information is obtained when cells of different origin are used. The formation of aggregates between neutrophils of an LAD patient and control neutrophils was thus quantified. Because neutrophil aggregation is dependent mostly on the presence of complement receptor type 3 (CR3), which is not present on LAD neutrophils, this result revealed the presence of a counter-structure for CR3 on LAD neutrophils (and hence on normal human neutrophils). In addition to the presence of these proteins on the cell surface, aggregation required continuous cell triggering as indicated by the transient aggregation induced by short-term activation of protein kinase C. This phenomenon was substantiated by the fact that energy depletion caused profound disaggregation. The present study reveals that neutrophil aggregation is a well-controlled process, which needs constant activation of CR3 for a stable interaction with a constitutively expressed counter-structure.

Adenosine Triphosphate↗

PubMed1995

Differential activation of c-fos promoter elements by serum, lysophosphatidic acid, G proteins and polypeptide growth factors.

The upstream regulatory region of the c-fos promoter contains two growth factor-regulated promoter elements: the serum response element, which binds a ternary complex comprising serum response factor (SRF) and a ternary complex factor (TCF); and the sis-inducible element (SIE) which binds STAT transcription factors. We used transient transfection of c-fos promoter mutants in NIH 3T3 cells to assess the contributions of these elements to activation by different extracellular stimuli. Colony-stimulating factor-1, platelet-derived growth factor and epidermal growth factor activate the c-fos promoter via cooperation of the SIE and the SRE; however, mutants that can bind SRF but not STATs or TCF remain inducible by whole serum. Activation by the SIE is context-dependent: interferons activate STAT DNA binding activity and transcription of SIE reporter genes, but not the c-fos promoter, which requires an additional ras-dependent signal. SRE activation by receptor tyrosine kinases requires TCF binding, and can be mediated by the TCF Elk-1. In contrast, SRE activation following activation of heterotrimeric G proteins by lysophosphatidic acid or aluminium fluoride ion requires SRF but is independent of TCF binding. These results suggest that heterotrimeric G proteins activate a signalling pathway distinct from those that activate the STATs and the TCFs, that controls SRF activity.

3T3 Cells↗

PubMed1983

Comparison of indirect probes of membrane potential utilized in studies of human neutrophils.

Four indirect probes of membrane potential, triphenylmethylphosphonium ion (TPMP+), 3,3'dipentyloxacarbocyanine [di-O-C5(3)], 3,3'dipentylindocarbocyanine [di-I-C5(3)], and 3,3'dipropylthiodicarbocyanine [di-S-C3(5)] have been used to study neutrophil (PMN) activation. The data extend previous studies indicating that the cyanine dye di-S-C3(5) not only exhibits a different fluorescence response mechanism from di-O-C5(3) [and di-I-C5(3)] but also that the fluorescence of di-S-C3(5) is destroyed by reactive oxygen products produced by neutrophils following stimulation. When these aspects of the probes are taken into account, the interpretations of the results using all three cyanine dyes are identical. Studies with the isotope TPMP+ indicate that long incubations are necessary for PMN to fully equilibrate during which time the PMNs depolarize. Use of TPB-, to shorten the TPMP+ equilibration time, produces results identical with those obtained using the cyanine dyes. The cyanine dyes and TPMP+/TPB- are toxic to neutrophil functions although they do not cause cell death. Toxicity can be avoided by using low concentrations of di-O-C5(3) and di-I-C5(3) but cannot be avoided with di-S-C3(5) or TPMP+/TPB-. Using di-O-C5(3) with the fluorescence-activated cell sorter, we demonstrate that heterogeneity of neutrophil responsiveness confuses the interpretation of studies characterizing the ionic basis of the fluorescence responses stimulated by certain stimuli. We conclude that some of the discrepancies currently reported in the literature using these probes are not due to inherent differences in the ability of the different probes to monitor the same event (i.e., PMN membrane potential) but instead are due to failure to correct for probe-specific problems or response heterogeneity.

Carbocyanines↗

PubMed2006

Cell senescence: hypertrophic arrest beyond the restriction point.

Withdrawal of mitogens (growth factors) arrests normal cells in G0 (quiescence). All other stresses and factors arrest cell cycle beyond the restriction point in G1 and G2 (non-G0 arrest), in the presence of mitogenic stimulation. Strong mitogenic stimuli by themselves cause non-G0 arrest. Unlike G0, arrest beyond restriction point is characterized by both high levels of cyclins and CDK inhibitors, activated mitogenic pathways with a secondary GF resistance, and continuous mass growth (cell hypertrophy). Prolonged hypertrophic arrest culminates in cell senescence. This review discusses that quiescence and senescence are two opposite, mutually exclusive conditions and that cell senescence can be reversed and prevented.

Aging↗

PubMed2002

Effects of Rosa canina fruit extract on neutrophil respiratory burst.

Respiratory burst leads polymorphonuclear neutrophils (PMN) to produce reactive oxygen species (ROS) such as superoxide anions (O(2)(o-)), hypochlorous acid (HOCl) and hydrogen peroxide (H(2)O(2)) which may possess deleterious effects for the organism. Rosa canina fruits are well known to contain a large amount of vitamin C which is antioxidant. This study was focused on the polyphenolics contained in rose hips to evaluate their antioxidative properties. We prepared a rose hip extract deprived of vitamin C. The extract contained mainly phenolics such as proanthocyanidins and flavonoids. We investigated its effects directly against (O(2)(o-)), HOCl and H(2)O(2) and investigated its effects on isolated PMN. For that, in vitro inflammatory conditions were reproduced by stimulating PMN with stimuli having different transductional pathways, in order to determine a possible mechanism of action. The results showed that the extract can inhibit ROS tested in acellular and cellular systems. The IC(50) obtained were 5.73 mg/L, 1.33 mg/L and 2.34 mg/L respectively for (O(2)(o-)), HOCl and H(2)O(2) in acellular experiments. For cellular experiments, the IC(50) were quite similar. Thus, the extract did not present an effect on PMN metabolism. Therefore, the antioxidative effects of Rosa canina are due not only to vitamin C but also to polyphenolics.

Anthocyanins↗

PubMed2002

Glucose transport by osmotic shock and vanadate is impaired by glucosamine.

In 3T3-L1 adipocytes, we previously reported that glucosamine impairs insulin stimulation of glucose transport, which is accompanied by impaired insulin stimulation of serine/threonine kinase Akt. To examine the role of Akt in glucosamine-induced insulin resistance, we investigated time course for insulin stimulation of Akt activity and glucose transport during recovery from glucosamine-induced insulin resistance. After induction of insulin resistance by glucosamine, we washed cells to remove glucosamine and incubated them for various times. After one hour, insulin stimulated-glucose transport was significantly increased and continued to increase up to 6-24 h. Insulin stimulation of Akt, however, did not increase after 1-3 h and began to slightly increase after 6 h. Next, we investigated effects of osmotic shock and vanadate on glucose transport in glucosamine-treated cells and found that glucosamine completely inhibited their actions in these cells. These data suggest that an Akt-independent mechanism is operative in glucosamine-induced insulin resistance and glucosamine impairs glucose transport stimulated by various stimuli involving and not involving Akt activation.

Adipocytes↗

PubMed1995

Signaling by a new anti-Thy 1 monoclonal antibody inhibits T cell proliferation and interferes with T-cell-mediated induction of costimulatory molecule B7-2.

Costimulatory activity on antigen-presenting cells is a critical determinant of the fate T cells when the T cell receptors are engaged by MHC:peptide complexes. Therefore, control of the expression of the costimulatory molecules regulates T cell responses. While several types of interactions between T cells and B cells up-regulate costimulatory molecules on antigen-presenting cell, no T cell surface molecules have been implicated in inhibiting the induction of the costimulatory molecules on B cells. Here we characterize a new anti-Thy1 mAb, 21F10, which inhibits T cell proliferation to selective stimuli. T cells stimulated by anti-CD3 together with anti-Thy1 mAb are anergic to further stimulation through the CD3, which suggests that the anti-Thy1 mAb interferes with the delivery of the costimulatory activity to T cells. Consistent with this notion, anti-Thy1 mAb 21F10 completely inhibits the induction of B7-2 on B cells. Induction of several T cell surface molecules such as CD69 and CD40 ligand was largely unaffected. As this inhibition requires a bivalent anti-Thy1 mAb and does not require binding of more than 50% of Thy1 molecules on T cell surface, we suggest that Thy1 may mediate a negative signaling pathway which inhibits the T-cell-mediated induction of costimulatory activity, including expression of co-stimulatory molecule B7-2.

Animals↗

PubMed1996

Release of sIL-2R alpha from and activation of native human peripheral blood mononuclear cells by recombinant IL-15.

The cytokine interleukin (IL)-15 shares several activities with IL-2. Both cytokines induced expression of cell-surface IL-2R alpha (CD25) on freshly isolated human peripheral blood mononuclear cells (PBMC) in the absence of other exogenous stimuli. They also stimulated the release of soluble IL-2R alpha and induced proliferation of these cells in 1-week cultures in a time- and dose-dependent manner. Recombinant IL-7 could also induce the expression of CD25, although sIL-2R alpha was released at only low levels. In monocyte-depleted PBMC the sIL-2R alpha release was minimal. When isolated T cells or non-T cells were stimulated by rIL-15 or rIL-2, cell surface CD25 was expressed, but released sIL-2R alpha was undetectable. On stimulation with rIL-15, more than 80% of all natural killer cells expressed CD25 and more CD8br+ lymphocytes were positive for CD25 compared to stimulation by rIL-2. These results may be clinically relevant because several diseases are associated with high serum levels of sIL-2R alpha which may he not only due to IL-2 but also due to IL-15 stimulation.

Cell Division↗

PubMed1996

Use of Blocking Antibodies as Probes for in Vivo Functions of Chemokines

Leukocyte infiltration into an inflammatory site is one of the pathological hallmarks of inflammatory reaction. Locally produced chemotactic factors are presumed to mediate the sequence of events leading to tissue injury associated with the infiltration of leukocytes. Chemotactic cytokines (chemokines) have been identified as being produced by various types of cells upon stimulation with inflammatory stimuli and exhibit a variety of effects on leukocytes in vitro and in vivo. Administration of highly specific neutralizing antibodies against these chemokines in several types of animal inflammation models clearly suggests important roles of these chemokines in recruiting and activating specific types of leukocytes at the inflammatory sites. Anti-IL-8 Ab treatment prevented neutrophil-dependent tissue damage as well as neutrophil infiltration in lipopolysaccharide (LPS)-induced dermatitis, LPS/IL-1-induced arthritis, lung reperfusion injury, and acute immune complex type glomerulonephritis in rabbits. Moreover, anti-MCP-1 Ab and anti-RANTES Ab inhibited macrophage infiltration in IgA immune complex alveolitis in rats and influx of lung macrophages in a murine model of endotoxemia, respectively. The use of anti-MIP-1alpha Ab also revealed that MIP-1alpha mediates eosinophil infiltration in allergic, granulomatous reactions in vivo.

Journal Article↗

PubMed2000

Costimulatory effects of interferon-gamma and interleukin-1beta or tumor necrosis factor alpha on the synthesis of Abeta1-40 and Abeta1-42 by human astrocytes.

Chronic inflammation and astrocytosis are characteristic histopathological features of Alzheimer's Disease (AD). Astrocytes are one of the predominant cell types in the brain. In AD they are activated and produce inflammatory components such as complement components, acute phase proteins, and cytokines. In this study we analyzed the effect of cytokines on the production of amyloid beta (Abeta) in the astrocytoma cell line U373 and in primary human astrocytes isolated postmortem from healthy aged persons as well as from patients with AD. Astrocytes did not produce Abeta in the absence of stimuli or following stimulation with IL-1beta, TNFalpha, IL-6, and TGF-beta1. Neither did combinations of TNFalpha and IL-1beta, IL-6 or TGF-beta1, or the coadministration of IFNgamma and IL-6 or TGF-beta1 induce Abeta production. In contrast, pronounced production of Abeta1-40 and Abeta1-42 was observed when primary astrocytes or astrocytoma cells were stimulated with combinations of IFNgamma and TNFalpha or IFNgamma and IL-1beta. Induction of Abeta production was accompanied by decreased glycosylation of APP as well as by increased secretion of APPsbeta. Our results suggest that astrocytes may be an important source of Abeta in the presence of certain combinations of inflammatory cytokines. IFNgamma in combination with TNFalpha or IL-1beta seems to trigger Abeta production by supporting beta-secretase cleavage of the immature APP molecule.

Aged↗

PubMed1990

Effect of local anaerobiosis on heart rate.

The isolated leg of a rat was connected to the body only by nerve and bone and was perfused with hypoxic Tyrode solution. Heart rate increased when metabolic parameters (PCO2, pH and lactate) reached values similar to those observed at the beginning of exercise. When the muscle was additionally stimulated by electric stimuli a significant temporal correlation between lactate and heart or respiratory rate was found. Metabolic changes caused by hypoxia and muscular contraction, in particular lactic acid, appear to act as chemical stimuli for metabolic muscle receptors participating in the generation of circulatory and respiratory responses to physical exercise.

Afferent Pathways↗

PubMed1986

Cerebellar Purkinje cell activity related to the classically conditioned nictitating membrane response.

Because of the purported critical role of cerebellar lobule HVI in classical conditioning of the nictitating membrane response of the rabbit, we recorded extracellularly from HVI Purkinje cells (PCs) during differential conditioning. Rabbits were trained using tonal conditioned stimuli (CSs) and stimulation of the periocular region as the unconditioned stimulus (US). Many PCs responded to the US, the most frequently observed response being a burst of simple spikes. PCs in HVI showed a variety of responses to CSs that were related to conditioned responses (CRs). The most frequently observed response was an increase in simple spikes correlated with CRs. The activity of many of these cells antedated CRs by 20-200 ms. A smaller proportion of cells exhibited inhibition of simple spike activity that antedated CRs. The existence of PCs that alter their firing before CRs suggests that they may be causally involved in this behavior, and in this respect they reinforce reports that lesions of HVI or its connections disrupt nictitating membrane CRs. Although complex spike activity was not generally related to the US or to CRs, a few PCs responded in relation to CRs with only complex spikes. In demonstrating CR-related activity in cerebellar PCs, this study supports theories of cerebellar learning such as those of Marr and Albus.

Action Potentials↗

PubMed1981

Tone-versus FM--induced patterns of excitation and suppression in the 14-C-2-deoxyglucose labeled auditory "cortex" of the guinea fowl.

The primary auditory "cortex" field L, of the Guinea fowl is a three layer tonotopically organized structure. Isofrequency planes as shown with the 2-deoxyglucose (2DG) method cut across these layers and with their second dimension extend in rostro-caudal direction. The input layer L2 exhibits "spontaneous" labeling due to high spontaneous activity of input terminals and units throughout the hearing range. The labeling is stronger locally along a rostro-caudal isofrequency contour of L2 after tone or narrow band FM stimulation. With tone stimuli the layers L1 and L3 are labeled within an isofrequency plane except for the rostral half of the field whereas frequency modulated tones do label these two layers throughout the corresponding isofrequency plane. FM stimuli in addition lead to a reduction of spontaneous labeling in frequency planes adjacent to those which are covered by the stimuli. Since these effects correlate with known inhibitory effects of such stimuli it is argued that the 2 DG method can identify the suppression of activity of neurons in suitable structures.

Animals↗

PubMed1990

Control of respiratory motor pattern by sensory neurons in spinal cord of lamprey.

Extracellular stimulation over the dorsal funiculus in the spinal cord of lampreys was found to selectively activate prolonged episodes of fictive arousal respiration. The induced episodes showed comparable increases in cycle frequency and motoneuron burst duration to the spontaneous arousal pattern observed in isolated brain preparations. Intracellular stimulation of primary sensory neurons with axons in the dorsal funiculus, called 'dorsal cells', also elicited the arousal pattern. Mechanoreceptive dorsal cells respond to cutaneous stimulation. When mechanical stimuli were applied to the skin of intact lampreys or to lampreys with ipsilateral vagotomy, arousal respiration was induced. Bilateral, but not unilateral, trigeminal lesion blocked dorsal cell induction of the arousal response. Spontaneous arousal respiration was recorded from intact, unrestrained lampreys. These results suggest that fictive arousal respiration is the in vitro correlate of natural arousal respiration in lampreys, and that one mechanism leading to arousal respiration may be the activity of sensory dorsal cells. A model for respiratory motor pattern switching in lamprey is proposed. The model suggests that the normal and arousal patterns are produced by separately engaging rostral or caudal pattern generators in the medulla, rather than by modifying one pattern generator.

Action Potentials↗

PubMed1982

The effect of different patterns of long-term stimulation on contractile properties and myosin light chains in rabbit fast muscles.

Fast rabbit skeletal muscles (tibialis anterior and extensor digitorum longus) were stimulated for 2-28 days by electrodes implanted in the vicinity of the peroneal nerve to produce maximal contractions at two different frequency patterns: that occurring naturally in nerves to slow muscles (10 Hz continuously) or three bursts of tetani (40 Hz) per minute, each 5s in duration. Both types of frequency produced muscles more resistant to fatigue during isometric twitch contractions, and led to a prolongation of contraction time greater and more consistent with 10 Hz than with 40 Hz. The twitch/tetanus ration was significantly higher in muscles stimulated at 10 Hz for 3-4 weeks but was not different from controls in muscles stimulated at 40 Hz. Both types of stimulation led to the appearance of myosin light chains characteristic of slow muscles. Muscles stimulated for 4 weeks at 40 Hz developed greater twitch tension per gram, and had significantly smaller cross-sectional area of myofibrils than control muscles. It is concluded that long-term electrical stimulation of fast muscles can affect some muscle contractile properties to resemble those of slow muscles irrespective of frequency of stimulation, provided the total number of stimuli is comparable, the duration of stimulation is long enough (minimum 2 weeks) and all motor units are activated.

Animals↗

PubMed1994

Rate-dependent prolongation of action potential duration in single ventricular myocytes obtained from hearts of rats with streptozotocin-induced chronic diabetes sustained for 30-32 weeks.

We examined the characteristics of the action potentials of single ventricular myocytes obtained from the hearts of rats with chronically-induced diabetes. Male Wistar rats were made diabetic by injecting streptozotocin (65 mg/kg) and 30-32 weeks later the hearts were excised and used for an electrophysiological study. Action potentials were recorded from isolated right ventricular myocytes by an electrode fabricated for patch clamp in the whole-cell recording configuration. The action potential durations (APDs) of steady state chronic diabetic rat myocytes were longer than those of age-matched normal rat myocytes at all levels of repolarization (APD25, APD50, APD75, and APD90). As the stimulation frequency was increased (0.2-2 Hz), the APDs were lengthened in both diabetic and normal rats, and the difference of APDs between the groups was greater when the stimulation frequency was higher. When we examined alterations of APDs under conditions of train stimulation (2Hz, 20 stimuli), (1) the APDs in both groups were prolonged, and (2) the degree of prolongation of APD was significantly greater and the rate of APD prolongation was significantly faster in myocytes from the diabetic rats. The prolongation of APD in these heart cells is probably secondary to alteration of the transient outward current Ito, and sheds light on repolarization abnormality in cases of diabetic cardiomyopathy.

Action Potentials↗