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PubMed1989

[Molecular mechanisms of muscle contraction].

Studying the mechanism of muscular contraction not even in one of the cases deriving from 50 sources could be obtained a myosin preparation free of phosphate. Therefore the aim of the authors was to elaborate methods which provided means for producing preparations of an endogenous P content that approached the P content of myosins of live sources. The phosphoryl groups of myosin may be released by incubating these preparations with F-actin. The actin attaches to the interaction domain of myosin and upon its effect P migrates within the myosin molecule from the site of phosphorylation to the interaction domain. At the interaction the myosin attaches to the actin through the phosphoryl group subsequently the myosin head turns from 90 degrees to 45 degrees consequently the actin-filament moves to a 12 mm distance towards the center of the sarcomer. When the myosin-actin-phosphoryl bridge ceases to be the P attaches transiently to the actin and the anorganic phosphate releases from the actin. The active center of myosin is filled again with the gamma phosphoryl group of ATP and the head returns simultaneously to the resting position of 90 degrees.

Animals↗
PubMed2006

Salutary effects of tachykinin receptor antagonists in a rat model of postoperative ileus.

BACKGROUND: Postoperative ileus (PI) is a common surgical complication treated mainly with supportive measures. Tachykinins control gastrointestinal motility and modulate somatic and visceral pain sensation; therefore, the effect of tachykinin receptor antagonists in a rat model of PI using NK(1-3) antagonists, SR140333, SR48968, and SR142801, was investigated. MATERIALS AND METHODS: Intestinal transit was measured as Evans blue migration after varied nociceptive stimuli: skin incision (SI), laparotomy (LAP), or laparotomy plus gut manipulation (L + M) in anesthetized rats. RESULTS: Diethyl ether anesthesia and SI did not influence the intestinal transit of the dye in comparison to untreated animals--UN: 61.17 +/- 5.47, 62.10 +/- 8.30, and 56.70 +/- 4.10 cm, respectively. In contrast LAP and L + M have significantly reduced intestinal motility to 26.40 +/- 2.07 and 9.70 +/- 1.15 cm, respectively. SR140333 (3-30 microg/kg), SR48968 (1-30 microg/kg), and SR142801 (3-10 microg/kg) reversed the additional inhibitory effects of gut manipulation subsequent to LAP dose-dependently, the dye transit returning with the use of the most effective antagonist doses up to 25.28 +/- 1.08, 21.70 +/- 0.19, and 25.0 +/- 1.34 cm. The combinations of submaximal doses of NK(1) and NK(3), NK(2) and NK(3) and NK(1), and NK(2) and NK(3) antagonists were not more effective than a single-agent regimen. On the other hand SR140333 and SR48968 (NK(1) + NK(2) antagonists) acted additively, the intestinal transit reaching 26.60 +/- 0.85 cm. SR140333, SR48968, and SR142801 have not affected the intestinal passage in UN rats or those undergoing SI or LAP. CONCLUSIONS: SR140333, SR48968, and SR142801 exert a salutary action on suppressed gut motility following surgical manipulation of the gut, the combination of NK(1) and NK(2) antagonists being most beneficial.

Anesthetics, Inhalation↗
PubMed1989

Non-random migration of CD4 and CD8 T cells: changes in the CD4: CD8 ratio and interleukin 2 responsiveness of efferent lymph cells following in vivo antigen challenge.

In this investigation we have examined some of the cellular and molecular changes in efferent lymph that drains from an antigen-stimulated peripheral lymph node. Resting efferent lymph is characterized by a higher percentage of CD4+ cells and consequently, a higher CD4/CD8 ratio than peripheral blood. Following antigen stimulation of a cannulated peripheral lymph node in antigen-primed sheep, the percentage of CD4+ cells in efferent lymph increases above the resting level during days 1, 2 and 3 post antigen stimulation. This is followed on days, 3, 4, and 5 after antigen stimulation by an increase in the percentage of CD8+ cells above the resting level which occurs as the percentage of CD4+ cells returns to the resting level. These changes cause the CD4/CD8 ratio to first increase above the resting value during the CD4 phase and then decrease below the resting value during the CD8 phase. During the CD4 phase a lymphokine activity is present in cell-free lymph fluid. Lymph fluid collected at this time supports the proliferation of activated T cells. Supernatants generated from efferent cells collected at a similar time and cultured in vivo for 24 h without any further stimulation are capable of releasing this material. During the CD8 phase cells expressing functional interleukin(IL)2 receptors appear in lymph fluid. The data suggests a sequential exit of T cell subsets from an antigen-stimulated lymph node and that the appearance of IL2-like activity and IL2-responsive cells in efferent lymph fluid are temporally distinct events.

Animals↗
PubMed2006

Temporal profile of neural stem cell proliferation in the subventricular zone after ischemia/hypoxia in the neonatal rat brain.

OBJECTIVES: Ischemia/hypoxia (I/H) causes severe neonatal brain injury, such as periventricular leukomaracia and hypoxic/ischemic encephalopathy. Neural stem cell research could lead to a treatment for such disorders. In order to elucidate the dynamic changes in neural stem cells in the neonatal brain after I/H, we investigated the proliferation of new cells in the subventricular zone (SVZ). METHODS: Seven-day-old Wister rats were subjected to ligation of the left carotid artery followed by 2 hours of hypoxic stress (8% O(2) and 92% N(2), at 33 degrees C). In order to elucidate the dynamic change of neural stem cells in the SVZ, single bromodeoxyuridine (BrdU; 50 mg/kg) was administered 2 hours before death 1, 7, 14 and 21 days after I/H. Immunohistochemical and immunofluorescent studies for BrdU and doublecortin (DCX) were carried out. As a control, a group of rats was subjected to sham surgery (incision of skin, but no ligation of the carotid artery) and no I/H. RESULTS: The numbers of BrdU-labeled cells in the SVZ, for both the ipsilateral side and the contralateral side of the I/H brain, were twice the level of the control at 7 days after I/H, but the numbers for both sides returned to the control level at 21 days. In the ipsilateral side of the I/H brain, the number of BrdU-labeled cells in the SVZb (lining the upper wall of lateral ventricle) was 4-fold at 7 days and 15-fold at 21 days after I/H compared with the control level. This chronological pattern is very similar to the pattern for I/H results of the posterior periventricle (pPV). DCX appeared in most BrdU-labeled cells in the SVZb and pPV. DISCUSSION: These findings indicate that I/H enhances neural stem cell proliferation in the SVZ, and some newborn cells migrate as neural precursors to the SVZb and pPV after I/H in the neonatal rat brain.

Animals↗
PubMed1976

In vitro stimulation of neutrophil motility by levamisole: maintenance of cgmp levels in chemotactically stimulated levamisole-treated neutrophils.

Levamisole at concentrations of 10(-3) M or 10(-4) M consistently increased neutrophil random motility and chemokinesis (stimulated random migration). Similar concentrations also increased directional movement of polymorphonuclear leukocytes to both endotoxin-activated serum and hydrolyzed casein. This effect on chemotaxis was due to a true stimulation and was not due solely to increased random movement. The effect of levamisole on the neutrophils could be removed by washing, but persisted if the cells were initially treated with levamisole and serum or endotoxin-activated serum. After neutrophil stimulation with chemotactic factor an initial rise in intracellular cyclic AMP levels was detected which was not influenced by prior levamisole treatment. Intracellular cyclic GMP levels after an initial slight depression, returned to resting levels and gradually diminished over a 60-minute period. Levamisole-treated cells consistently showed higher cyclic GMP levels and it is postulated that by maintaining intracellular cyclic GMP levels, microtubular assembly and cell motility might be enhanced.

Caseins↗
PubMed1989

Electron transfer process in cytochrome oxidase after pulse radiolysis.

The reduction of bovine heart cytochrome oxidase by the 1-methylnicotinamide (MNA) radical was investigated by the use of pulse radiolysis. With the decay of the MNA radical, the absorption at 445 and 605 nm, a characteristic to ferrous heme a of the oxidase, increased. The kinetic difference spectrum obtained was similar to that of the fully reduced minus the fully oxidized form of the oxidase, and was not different from that obtained in the reaction of the MNA radical with the mixed valence CO complex of the oxidase, where heme a3 is the CO-bound reduced form with heme a oxidized. This suggests that the absorption changes at 445 and 605 nm arise from the reduction of heme a, not heme a3. In order to elucidate the contribution of "visible" copper in this reaction, the absorption of the oxidase in the near-infrared region was measured. A decrease of the 830 nm band due to the reduction of visible copper was detected with a half-life of 5 microseconds. This absorption change obeyed pseudo-first order kinetics and its rate constant increased with the concentration of the oxidase. This suggests that the absorption change at 830 nm is followed by a bimolecular reaction of the MNA radical with visible copper of the oxidase. After the first phase of the reduction, the return of the 830 nm band corresponding to oxidation of the copper was observed with a half-life of 100 microseconds. Concomitantly, the absorption at 605 and 445 nm due to the reduction of heme a increased. The rates of oxidation of the copper were identical to those of the reduction of heme a and independent of the oxidase concentration. This suggests that the MNA radical reacts with visible copper of the oxidase with a second order rate constant of 1.5 X 10(9) m-1 s-1 and subsequently the electron flows to heme a by intramolecular electron migration with a first order rate constant of 1.8 X 10(4) s-1. An activation energy of the intramolecular electron transfer was calculated to be 2.8 kcal/mol in the range 4-33 degrees C.

Animals↗
PubMed1992

The role of myoneural and luminal continuity in the coordination of canine gastroduodenal patterns of motility.

Our aim was to determine the role of intrinsic myoneural and enteric luminal continuity in the coordination of gastric and duodenal motility patterns. Three groups of dogs were prepared: five dogs with an intact gastrointestinal tract served as a Control group; four dogs had transection and reanastomosis of the duodenum 0.5 cm distal to the pylorus (Pyloric Transection group); and seven dogs had identical proximal duodenal transection, but with oversewing of duodenum and pylorojejunostomy to a Roux-en-Y limb (Roux-en-Y group). In the Control and Pyloric Transection groups, the gastric and intestinal MMCs were similar in appearance, the cycle durations (x +/- SEM) were not different (134 +/- 19 vs 111 +/- 26 min, respectively; P > 0.05), and the times between the start of gastric and duodenal Phase III (gastroduodenal latency) were similar (6 +/- 1 vs 10 +/- 3 min; P > 0.05). In the Roux-en-Y group, MMCs also occurred in six of seven dogs but tended to have a longer cycle duration (176 +/- 19 min) and a more variable gastroduodenal latency (23 +/- 15 min). Plasma motilin concentration, measured only in the Roux-en-Y group, was greater during Phase III in the stomach and duodenum than during Phases I or II (P < 0.05). Feeding inhibited the gastric and duodenal MMCs in all groups, but the duodenal MMC returned earlier in the Roux-en-Y group. The Roux-en-Y jejunal limb exhibited a postprandial pattern in only seven of 14 studies.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1983

Isolation, purification, and characterization of deer mouse (Peromyscus maniculatus bairdii) prolactin.

Prolactin (PRL) secreted by Peromyscus maniculatus bairdii anterior pituitaries was purified by gel filtration on Sephadex G-100 and ion-exchange chromatography on DEAE-cellulose. Peromyscus PRL (pmPRL) eluted from Sephadex G-100 with an elution-to-void volume ratio of 1.9 and at a salt concentration of 100 mM NaCl on DEAE-cellulose. Electrophoretic homogeneity of the hormone was demonstrated in several gel systems. On 7 1/2% alkaline polyacrylamide gels, pmPRL migrated with an Rf of 0.65. The molecular weight of pmPRL was estimated at 24,000 to 26,000 by sodium dodecyl sulfate electrophoresis and molecular exclusion chromatography. The amino acid composition of pmPRL was similar to other mammalian PRLs including two tryptophan residues and three disulfide bonds. Leucine was found to be the NH2-terminal residue. Circular dichroism (CD) spectra indicated an alpha-helix content of 55 +/- 5%, a value typical for prolactin molecules. However, in the region of side-chain absorption, the CD spectrum displayed a weak, asymmetric, negative band near 299 nm, with the CD returning to slightly positive values at 292 nm. This CD pattern is not typical of other secreted or stored prolactins. In the pigeon crop-sac assay, pmPRL showed a prolactin-like activity of 21 IU/mg.

Amino Acids↗
PubMed1997

Radiographic patterns and response to corticosteroid hip injection.

OBJECTIVES: A prospective, open study of corticosteroid hip injection (CHI) was performed to determine if different radiological patterns of arthritis vary in their response. METHODS: Forty five patients (15 with rheumatoid arthritis, 27 with osteoarthritis, and three with anklyosing spondylitis) underwent hip injection with 80 mg methylprednisolone and lignocaine under x ray control. Outcome was assessed at two, 12, and 26 weeks for pain, range of hip movement, and graded functional questionnaire. Patients estimated their pain in four components, night pain, rest pain, weight bearing, and referred pain, each measured by 10 cm visual analogue score and summed to give a total score out of 40 cm. Hip radiographs were evaluated blindly for pattern and severity of arthritis, as well as for progression between 0 and 26 weeks. RESULTS: Median total pain score decreased from 29 cm at baseline to 22 cm at two weeks (p = 0.0001), 24 cm at 12 weeks (p = 0.03), but had returned nearly to baseline by 26 weeks (25 cm, p = 0.3). Greatest improvement was seen for night pain. Mean range of internal rotation increased from 16 to 28 degrees at two weeks (p = 0.03) and 21 degrees at 12 weeks (p = 0.06). Functional ability did not change. Hips with an atrophic pattern of arthritis on plain radiography gained negligible pain relief at two weeks compared with hips with a hypertrophic or mixed bone response (p = 0.04). The degree of pain relief was similar in patients with OA and RA, and was not influenced by radiographic severity or by the direction of migration of the femoral head. CONCLUSION: Pain and internal rotation improve for up to 12 weeks after CHI. CHI offers a useful and safe therapeutic option for patients with hip arthritis, with the exception of those with a purely atrophic radiological pattern.

Adult↗
PubMed2000

Open-sea migration of magnetically disturbed sea turtles.

Green turtles (Chelonia mydas) that shuttle between their Brazilian feeding grounds and nesting beaches at Ascension Island in the middle of the Atlantic Ocean are a paradigmatic case of long-distance oceanic migrants. It has been suggested that they calculate their position and the direction of their target areas by using the inclination and intensity of the earth's magnetic field. To test this hypothesis, we tracked, by satellite, green turtles during their postnesting migration from Ascension Island to the Brazilian coast more than 2000 km away. Seven turtles were each fitted with six powerful static magnets attached in such a way as to produce variable artificial fields around the turtle that made reliance on a geomagnetic map impossible. The reconstructed courses were very similar to those of eight turtles without magnets that were tracked over the same period and in the previous year, and no differences between magnetically disrupted and untreated turtles were found as regards navigational performance and course straightness. These findings show that magnetic cues are not essential to turtles making the return trip to the Brazilian coast. The navigational mechanisms used by these turtles remain enigmatic.

Animals↗
PubMed1984

Complement-dependent killing of Nippostrongylus brasiliensis infective larvae by rat alveolar macrophages.

Histopathological studies have provided circumstantial evidence that helminth parasite destruction occurs in the lung; however controlled in vitro studies on the helminthocidal activity of lung cells have not been reported. This study presents evidence that Nippostrongylus brasiliensis infection in the rat induces alterations in broncho-alveolar lavage (BAL) cell numbers, differential counts, and in vitro helminthocidal activity. Normal, uninfected rats yielded 3.3 +/- 0.6 X 10(6) BAL cells/rat, consisting predominantly of alveolar macrophages (greater than 90%). However on days 2-8 post-infection there was a 1.5-2.4-fold increase in BAL cell numbers with a significant neutrophilia on day 2 and a significant increase in the absolute number of all cell types on day 8. On day 32 post-infection, BAL cell numbers had returned to control levels. Normal BAL cells neither adhered to nor killed N. brasiliensis infective larvae (L3) in the presence of rat complement. By contrast BAL cells recovered from infected rats on days, 2, 8 or 32 post-infection (D2, D8 and D32 BAL cells, respectively) adhered under similar conditions. However, only D8 and D32 BAL cells killed L3. This complement-dependent killing correlated with significantly increased numbers of C3 receptor bearing alveolar macrophages in D8 and D32 BAL cells. Complement-dependent alveolar macrophage helminthocidal activity may therefore play an important role in lung resistance against resident or migrating helminths.

Animals↗
PubMed1996

Effects of dexamethasone on bovine circulating T lymphocyte populations.

In cattle, gamma delta T cells represent a higher proportion of circulating T cells than in humans. Bovine gamma delta T cells can be recognized by expression of gamma delta cell receptor (gamma delta TCR) determinants or by a 215/230-kDa surface antigen (WC1). WC1 is expressed on 90% or more of circulating bovine gamma delta T cells. The effects of dexamethasone on this and other subsets (CD3, CD2, CD4, CD8) of peripheral blood T lymphocytes were determined by flow cytometric analysis. Twelve 15-month old bulls were injected with dexamethasone (0.04 mg/kg/day) for 3 consecutive days and four bulls were untreated controls. Blood samples were collected daily for 3 days before dexamethasone injections and for an additional 7 days starting on the third day. Data were recorded as percent positive cells and as mean fluorescent intensity (MFI) of positive cells. Initially, CD3+ cells represented 65-73% of all peripheral blood clear cells (PBMC). Dexamethasone reduced CD3+/- cells (PBMC). Dexamethasone reduced CD3+ cells to 30% and these recovered to 50% positive cells by 9 days after the last dexamethasone injection. Loss of CD3+ cells was not due to reductions in alpha beta T cells because dexamethasone did not influence the percent CD2+, CD4+, or CD8+ cells. However, percent WC1+ cells rapidly declined from a baseline of 26.4% of PBMC to < 6% by the final injection. During injections, the MFI of WC1 increased. The MFI of WC1 returned to control values 7 days after the last injection of dexamethasone, but the percent gamma delta T cells recovered to only 14% WC1+ PBMC by the final day of the study. During its maximum effects on WC1, dexamethasone also caused a profound decrease of L-selectin MFI on remaining PBMC (mostly alpha beta T cells and monocyte/macrophages). In a second trial, two-color analyses determined that dexamethasone did not increase apoptosis in WC1+ cells and did not reduce L-selectin MFI on either CD2+ of WC1+ cells. The cumulative results suggested that dexamethasone promoted gamma delta T cell migration out of peripheral blood via an L-selectin-independent mechanism and that dexamethasone did not alter alpha beta T cell migration kinetics.

Animals↗
PubMed2006

Shifting Wallgraft position: case reports and review of the forces affecting Wallgraft positioning.

Extensive use of stent-grafts in the iliac arteries is an increasingly common endovascular alternative to an aortobifemoral bypass procedure. The treatment of diffuse bilateral disease is frequently performed by using the technique of paired stent-grafts in the proximal common iliac arteries to effectively re-create the aortic-bifurcation. We present three patients treated with this technique who returned with recurrent occlusive disease. During subsequent treatment, the stent-grafts were noted to be in different positions from where they were initially deployed. A discussion of the possible explanations for the shifting positions follows.

Aged↗
PubMed2006

Dynamic changes in Robo2 and Slit1 expression in adult rat dorsal root ganglion and sciatic nerve after peripheral and central axonal injury.

Robos are transmembrane receptors that mediate Slit signaling to repel growth cone outgrowth and neural migration in the developing central nervous system. Their distribution and function in the peripheral nervous system remains unclear. In the present study, we examined expression of Slit1 and Robo2 in adult rat dorsal root ganglion (DRG), spinal cord and sciatic nerve after peripheral nerve injury (axotomy). In control rats, Slit1 and Robo2 mRNA and protein were expressed at basic levels in the L5 and L6 DRGs. Sciatic transection resulted in a significant up-regulation of both Robo2 and Slit1 mRNA and protein (p<0.05 versus control). The peak of Slit1 and Robo2 expression occurred at days 7 and 14, respectively, and returned to control levels at days 28 and 21 post-axotomy, respectively. By contrast, injury to the central axons of the DRG by dorsal rhizotomy did not up-regulate Slit1 and Robo2 expression. Robo2 staining was stronger in small diameter neurons than in large diameter neurons in control DRG. Interestingly, post-axotomy, Robo2 immunostaining increased in the large diameter neurons and the number of Robo2 positive large diameter neurons increased significantly relative to controls. Non-neuronal cells surrounding the primary sensory neurons, including the satellite cells, were Slit1-positive, and Slit1 protein was expressed in the myelin sheath and non-neural cells in both intact and degenerating sciatic nerve axons. Sciatic nerve transection also led to an accumulation of Slit1 protein in peripheral region of the traumatic neuroma. In conclusion, we report an altered expression and redistribution of Robo2 and Slit1 in the DRG and sciatic nerve trunk after peripheral axotomy. Our results indicate that Slit1 and Robo2 likely play an important role in regeneration after peripheral nerve injury.

Analysis of Variance↗
PubMed2002

Impairment of cytoskeleton-dependent vesicle and organelle translocation in green algae: combined use of a microfocused infrared laser as microbeam and optical tweezers.

A Nd-YAG laser at 1064 nm is used as optical tweezers to move intracellular objects and a laser microbeam to cause impairment of cytoskeleton tracks and influence intracellular motions in desmidiaceaen green algae. Naturally occurring migrations of large nuclei are inhibited in Micrasterias denticulata and Pleurenterium tumidum when the responsible microtubules are targeted with a laser microbeam generating 180 mW power in the focal plane. Impairment of the microtubule tracks appears to be irreversible, as the nucleus cannot pass the former irradiated area in Pleurenterium or remains abnormally dislocated in Micrasterias. The actin filament-dependent movement of secretory vesicles and smaller particles can be manipulated by the same IR-laser at 90 mW when functioning as optical tweezers. In Closterium lunula particles are displaced from their cytoplasmic tracks for up to 10 micro m but return to their tracks immediately after removing the light pressure gained by the optical tweezers. The cytoplasmic tracks consist of actin filament cables running parallel to the longitudinal axis of Closterium cells as depicted by Alexa phalloidin staining and confocal laser scanning microscopy. Dynamics and extensibility of the cytoplasmic strands connecting particles to the tracks are also demonstrated in the area of large vacuoles which are surrounded by actin filament bundles. In Micrasterias trapping of secretory vesicles by the optical tweezers causes irreversible malformations of the cell shape. The vesicle accumulation itself dissipates within 30 s after removing the optical tweezers, also indicating reversibility of the effects induced, in the case of actin filament-mediated processes.

Cell Nucleus↗
PubMedDate not supplied

Compartmentalisation between gut and lung mucosae in a model of secondary immunodeficiency: effect of thymomodulin.

UNLABELLED: Compartmentalisation of mucosal immune response seems to be the result mainly of the preferential migration of activated cells back to their inductive sites. The aim of this report was to demonstrate, in a model of secondary immunodeficiency in Wistar rats (severely protein deprived at weaning and refed with casein 20%; group R21), that the oral administration of Thymomodulin (group:R21TmB) has different effects on gut and BALT (Bronchus-associated lymphoid tissue). Tissue sections (5 mu) were studied by immunohistochemistry 1). The oral administration of Thymomodulin restores only in gut Lamina propria (LP) the IgA B and CD4 T cell populations to control levels. The CD8a and CD25 subpopulations do not vary in gut as they return to control levels when refed with 20% casein diet. All the populations mentioned above remained decreased even after receiving Thymomodulin by the oral route. However, the same behaviour was observed for the TCR delta T cells that were decreased and return to normal levels in both mucosae by the effect of the immunomodulator; 2) when studying the iIEL (intestinal intraepithelial lymphocytes) CD8 alpha, CD25 and TCR gamma delta T cells, that were increased in R21, return to control levels in R21TmB. In BALT intraepithelium CD8 alpha and CD25 T cells remained decreased, while only TCR gamma delta T cells (increased in R21) return to control values. CONCLUSIONS: 1) there exists a compartmentalisation between both mucosae, as T CD4+ and IgA B+ cells are restored by TmB only in gut; 2) only those iIEL involved in inflammation (CD8 alpha+/CD25+ and TCR gamma delta+/CD25+) are normalised by means of the Thymomodulin 3) however, in BALT,only TCR gamma delta+ T cells are restored 4) the oral administration of the present immunomodulator may be useful as a therapeutic agent, although the preferential survival in the tissue of initial stimulation is the major factor in the preferential distribution of activated cells.

Adjuvants, Immunologic↗
PubMed2005

5-Fluorouracil interferes with actin organization, stress fiber formation and cell migration in corneal endothelial cells during wound repair along the natural basement membrane.

Corneal endothelial cells respond to a circular freeze wound by undergoing actin cytoskeletal reorganization that is mainly characterized by the disappearance of circumferential microfilament bundles (CMBs) and the subsequent appearance of distinct stress fibers. This cytoskeletal rearrangement is associated with changes in cell shape as migrating cells lose their polyhedral appearance, spread out, and assume a stellate morphology with cell processes extending outward into the injured area. We report here that in the presence of low concentrations (0.01-0.l mM) of the anti-metabolite 5-fluorouracil (5-FU), characteristic actin organization becomes disrupted and migrating cells do not display elongated processes typical of control tissues and translocation into the injury zone is retarded, but not inhibited. Rhodamine phalloidin staining revealed no evidence of stress fiber formation. A higher concentration of 5-FU (1.0 mM) not only prevented formation of discernible stress fibers but also resulted in a more restricted cell movement during wound repair. That this was not a cytotoxic effect was demonstrated by transferring tissues back into standard medium allowing endothelia to reinitiate migration and undergo complete wound healing by 72 h post-transfer. Overnight incubation of endothelia in 4 muM phallacidin resulted in limited CMB disruption the extent of which was dependent on the 5-FU concentration. The effects of 5-FU on the actin cytoskeleton are reversible and by 24 h after placing treated endothelia into medium without 5-FU, actin begins to become re-established and by 48 h microfilament patterns in the tissue resemble those of non-treated endothelia. Similarly, when non-injured tissues are cultured in the presence of 5-FU for 24 h, subsequently injured and returned to standard medium, they exhibit no stress fibers when observed at 24 h post-wounding. However, by 48 h post-injury these cells now display stress fibers and extend processes into the wound area. Biochemical studies on isolated muscle actin demonstrated that actin polymerization is unaffected in the presence of either 0.01 or 1 mM 5-FU as determined by the F-actin sedimentation and falling ball viscosity techniques. Thus, the mechanism(s) by which 5-FU exerts its actions on the actin cytoskeleton appears to be one of an indirect nature.

Actins↗
PubMed1992

Ultrastructural, biochemical and electrophysiological changes induced by 5,6-dihydroxytryptamine in the CNS of the snail Helix pomatia L.

The serotonin neurotoxin, 5,6-dihydroxytryptamine (5,6-DHT), was injected into the body cavity of snails. Changes induced in the central nervous system (CNS) by the neurotoxin were studied by morphological, electrophysiological and biochemical techniques for up to 90 days following injection. The neurotoxin induced a variety of ultrastructural alterations during the early phase (1st to 6th days) following treatment. On day 6 after treatment, membranous structures first appeared in the synaptic-like areas and apparently migrated to cell bodies where they were detected by day 14. Their number increased with time. Neurotoxin-induced structural alterations were found in neuronal processes and cell bodies of the serotonergic metacerebral giant cells injected intracellularly with horseradish peroxidase and in serotonin immunoreactive axons. These findings suggest that the toxin-induced alterations are rather selective for the serotonin-containing neuronal elements. The neurotoxin decreased the concentration of 5-HT in and [3H]5-HT uptake into cerebral and pedal ganglia, with a maximum effect between the 3rd and 5th day following drug administration. 5-HT levels and 5-HT uptake returned to normal by 19-21 days after treatment. The concentration of dopamine and of [3H]DA uptake capacity were reduced between 3-5 days after injection of 5,6-DHT by 6-7 days following treatment. The transmission from identified serotonergic synapses to targets was reduced beyond day 5 after 5,6-DHT administration. By 15 days after treatment, synaptic transmission between the metacerebral giant cell (MGC) and buccal followers was blocked. Transmission recovered by day 21 after 5,6-DHT. Comparison of the time-course of functional and structural recovery indicates that while functional recovery takes place within 21 days after treatment, certain structural alterations, e.g. the membranous structures and dense particles, remain in the nerve fibres and cell bodies. These may serve as specific intracellular markers of the serotonin-containing neuronal elements long after functional recovery from the effect of 5,6-DHT.

5,6-Dihydroxytryptamine↗