Intact Tail Research Articles

Viability and acrosome staining of stallion spermatozoa by Chicago sky blue and Giemsa

A simple trypan blue-neutral red-Giemsa staining procedure for simultaneous evaluation of acrosome, sperm head, and tail membrane integrity and morphology has been used to evaluate equine spermatozoa. Some special characteristics and problems have arisen in evaluating stallion semen. One problem was the differentiation of intact vs. damaged sperm tails primarily in frozen and thawed samples. After freezing and thawing, a high percentage of spermatozoa with an unstained head and stained tail were observed. These cells are considered immotile. Therefore, unambiguous differentiation of intact vs. damaged sperm tail membrane is very important for evaluating semen quality. The aim of our study was to develop a method especially for stallion sperm to distinguish more accurately the different cell types. We compared Chicago sky blue 6B (CSB) to trypan blue (TB) for viability staining. CSB/Giemsa staining showed good repeatability and agreement with TB/Giemsa measurements. For densitometry analysis, individual digital images were taken from smears stained by CSB/Giemsa and by TB/Giemsa. A red-green-blue (RGB) histogram for each area of spermatozoa was drawn. Differences of means of RGB values of live vs. dead tails and separate live vs. dead heads from each photo were used to compare the two staining procedures. CSB produced similar live/dead sperm head differentiation and better tail differentiation. TB can be replaced by CSB and this results in more reliable evaluation. After staining with 0.16% CSB and 4 min fixation, 2–4 h Giemsa staining at 25–40° C is recommended for stallion semen.

Dependence of Mechanical Behavior of the Murine Tail Disc on Regional Material Properties: A Parametric Finite Element Study

In vivo rodent tail models are becoming more widely used for exploring the role of mechanical loading on the initiation and progression of intervertebral disc degeneration. Historically, finite element models (FEMs) have been useful for predicting disc mechanics in humans. However, differences in geometry and tissue properties may limit the predictive utility of these models for rodent discs. Clearly, models that are specific for rodent tail discs and accurately simulate the disc's transient mechanical behavior would serve as important tools for clarifying disc mechanics in these animal models. An FEM was developed based on the structure, geometry, and scale of the mouse tail disc. Importantly, two sources of time-dependent mechanical behavior were incorporated: viscoelasticity of the matrix, and fluid permeation. In addition, a novel strain-dependent swelling pressure was implemented through the introduction of a dilatational stress in nuclear elements. The model was then validated against data from quasi-static tension-compression and compressive creep experiments performed previously using mouse tail discs. Finally, sensitivity analyses were performed in which material parameters of each disc subregion were individually varied. During disc compression, matrix consolidation was observed to occur preferentially at the periphery of the nucleus pulposus. Sensitivity analyses revealed that disc mechanics was greatly influenced by changes in nucleus pulposus material properties, but rather insensitive to variations in any of the endplate properties. Moreover, three key features of the model-nuclear swelling pressure, lamellar collagen viscoelasticity, and interstitial fluid permeation-were found to be critical for accurate simulation of disc mechanics. In particular, collagen viscoelasticity dominated the transient behavior of the disc during the initial 2200 s of creep loading, while fluid permeation governed disc deformation thereafter. The FEM developed in this study exhibited excellent agreement with transient creep behavior of intact mouse tail motion segments. Notably, the model was able to produce spatial variations in nucleus pulposus matrix consolidation that are consistent with previous observations in nuclear cell morphology made in mouse discs using confocal microscopy. Results of this study emphasize the need for including nucleus swelling pressure, collagen viscoelasticity, and fluid permeation when simulating transient changes in matrix and fluid stress/strain. Sensitivity analyses suggest that further characterization of nucleus pulposus material properties should be pursued, due to its significance in steady-state and transient disc mechanical response.

Gene expression profiling in formalin-fixed, paraffin-embedded (FFPE) benign and cancerous prostate tissues using universal bead arrays

9526 Background: Archival tissues from patients with known clinical history have become an invaluable resource for gene expression analysis for tumor classification and clinical outcome prediction. These samples may be studied using qPCR, but RNA extracted from FFPE tissues is often significantly degraded, making them difficult to use in microarray analyses. Methods: We have developed the DASL assay, a flexible, sensitive and reproducible expression profiling method for parallel analysis of hundreds of genes in FFPE samples using universal bead arrays. The method can tolerate partial RNA degradation because it does not depend on an intact poly-A tail for cDNA synthesis. Tissue- and cancer-specific expression profiles have been obtained with as little as 50 ng total RNA derived from FFPE tissues that had been stored up to 11 years (Bibikova, et al., Am. J. Pathol. 165:1790–1807, 2004). Results: We performed a profiling study of 174 benign or cancerous prostate FFPE tissues retrieved from the VA San Diego Healthcare System. A total of 512 prostate cancer-related genes selected from the literature were monitored at 3 probes per gene. Despite RNA degradation, highly reproducible expression profiles were obtained for these samples (average r2 = 0.98). Expression data were used to identify tumor- and stroma-specific genes, which were then included in a sample classification scheme. Resulting gene panels correlated with clinical parameters such as tumor type, tumor grade, therapeutic response, and patient survival. In additional studies using laser capture microdissection of FFPE tissues, increased expression of tumor- and stroma-specific genes were measured in the correct cell types, confirming the results based on heterogeneous tissues. Conclusions: Putative prostate cancer biomarkers were identified using the DASL expression assay and RNAs derived from FFPE patient samples. In combination with the high-throughput capability of the bead array-based platform, the DASL assay may be used to monitor hundreds of genes in hundreds of samples at once. These data suggest that FFPE tissue archives, with known patient outcomes, can now be accessed for microarray expression analysis. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Illumina Illumina

319 USE OF PENTOXIFYLLINE AND HYALURONIC ACID FOR STALLION SPERM SEPARATION

In this study, we reduced the volume of separating media and the time of centrifugation to increase the yield of viable sperm for ICSI when few sperm are available. Hyaluronic acid (HA) was used successfully in combination with swim-up for separating bull spermatozoa (Shamsuddin and Rodriguez-Martinez 1994 Anim. Reprod. Sci. 36, 61–75). The purpose of this study was to compare the effectiveness of mini-Percoll and swim-up for low numbers of sperm treated or non-treated with HA or pentoxifylline (PX), a molecule that stimulates sperm motility (Gradil and Ball 2000 Theriogenology 54, 1041–1047). Poor-to-medium quality frozen semen (0.5-mL straws, 200 million cells/mL) from 3 stallions was used 3 times each. Two straws were thawed and mixed. Aliquots of 100 μL semen were allocated to 7 treatments. Three aliquots were incubated at 38°C for 20 min before Percoll-centrifugation (P-NT: non-treated; P-PX: 3.5 mM PX; or P-HA: 1 mg/mL HA, each in 0.25 mL chemically defined handling medium (HCDM)); one aliquot was centrifuged through Percoll without incubation (P-CON: control). Our mini-Percoll was a two-layer gradient (0.4 mL 90% and 0.5 mL 45% Percoll) in a 1.5-mL microcentrifuge tube that was centrifuged at 600g for 5 min. Then a 30-μL pellet was washed in HCDM at 300g for 5 min. At the same time, 3 aliquots of 100 μL sperm were placed in 1 mL HCDM for swim-up (S-NT: non-treated; S-PX: 3.5 mM PX; or S-HA: 1 mg/mL HA) for 30 min. A 0.65-mL aliquot of supernatant was centrifuged in HCDM at 300g for 5 min. In every treatment, the final 30-μL pellet aspirated from the bottom was evaluated. Recovery rate was determined using a hemacytometer. Sperm head, tail and acrosome membrane integrity were evaluated with Kovacs-Foote staining (Kovacs and Foote 1992 Biotech. Histochem. 67, 119–124) counting 300 sperm/sample. Cells were classified into 5 categories: intact head, tail and acrosome membrane (Intact); intact head, tail, damaged acrosome (IHITDA); intact head, damaged tail; damaged head, intact tail; damaged head, tail, acrosome. P-CON and P-PX resulted in more intact sperm compared to all swim-ups. IHITDA was nonsignificantly (P > 0.05) higher in P-PX than in P-CON (17 vs. 11%). Better recovery rates were found in P-CON, P-PX, and S-HA than in P-NT, P-HA, S-NT and S-PX. Hyaluronic acid increased recovery rate during swim-up, but not viability in any of the treatments. Pentoxifylline is beneficial if Percoll separation is delayed, but clarification of its effect on acrosome exocytosis is needed. Table 1. Intact and recovery rates (LS means ± SE)

Reevaluation of the virulence of prototypic strain 15 of pneumonia virus of mice.

Prototypic strain 15 of pneumonia virus of mice (PVM) has been described as being nonpathogenic in mice, in contrast to the mouse-passaged, highly virulent strain J3666. Previous sequence analysis also indicated that strain 15 encodes an attachment G protein that is truncated at the amino terminus, which for the amino terminally anchored protein deletes the cytoplasmic tail. However, we found that PVM strain 15 obtained from the American Type Culture Collection was highly virulent in mice and was essentially indistinguishable on that basis from strain J3666. Sequence analysis showed that this preparation of virus encodes a G protein with an intact cytoplasmic tail: the truncated predicted protein in the previous sequence appeared to be due to a single nucleotide insertion that disrupted the upstream end of the open reading frame and shifted the translational start site to the next downstream AUG. Taken together, the two studies indicate that strain 15 is an inherently virulent strain but that a nonpathogenic variant that was generated during passage in vitro and encodes a truncated G protein exists. Interestingly, the majority sequence of strain J3666 was found to encode a G protein with an extended cytoplasmic tail, suggesting that there is the potential for considerable plasticity in the cytoplasmic tail of the G protein of PVM.

Quantitative Gene Expression Profiling in Formalin-Fixed, Paraffin-Embedded Tissues Using Universal Bead Arrays

We recently developed a sensitive and flexible gene expression profiling system that is not dependent on an intact poly-A tail and showed that it could be used to analyze degraded RNA samples. We hypothesized that the DASL (cDNA-mediated annealing, selection, extension and ligation) assay might be suitable for the analysis of formalin-fixed, paraffin-embedded tissues, an important source of archival tissue material. We now show that, using the DASL assay system, highly reproducible tissue- and cancer-specific gene expression profiles can be obtained with as little as 50 ng of total RNA isolated from formalin-fixed tissues that had been stored from 1 to over 10 years. Further, tissue- and cancer-specific markers derived from previous genome-wide expression profiling studies of fresh-frozen samples were validated in the formalin-fixed samples. The DASL assay system should prove useful for high-throughput expression profiling of archived clinical samples.

Factors affecting fertilization of porcine oocytes following intracytoplasmic injection of sperm

The objective of this study was to optimize intracytoplasmic sperm injection (ICSI), and to assess the effects of membrane-damaged sperm on development of porcine oocytes following ICSI. For optimization of development following the ICSI process, sperm injected oocytes were activated 0.5-1.0 hr after ICSI with 1 x 30 micros pulse of 1.2, 1.7, 2.2, and 2.7 kV/cm DC in experiment 1. After 7-days of culture ICSI oocytes activated with [x1]2.2 kV/cm produced more blastocyts ([x2]34.4%, P < 0.05) than other treatment groups. In experiment 2, oocytes were activated with 1 x 30 micros pulse of 2.2 kV/cm at either 0, 1.5, 3, or 6 hr after ICSI. Oocytes activated 1.5 hr after [x3]ICSI yielded more blastocysts (27.6%)[M4] than in other treatments. In experiment 3, sperm were briefly exposed to 0.1% Triton X-100 to induce membrane damage. Live-dead staining of Percoll-sorted untreated spermatozoa (frozen-thawed) used in this study showed that over 96% were "alive" whereas none were "alive" after Triton X-100 treatment. The rate of development to blastocyst of oocytes injected with Triton X-100 treated sperm combined with electrical activation (EA) at 1 x 30 micros pulse of 2.2 kV/cm (EA, 40.0%) was the best, when compared with those injected with untreated sperm plus EA (P < 0.05). In experiment 4, the development rate of oocytes to the blastocyst stage ([x5]32.1%) following injection of a sperm head only was not significantly different from that of oocytes injected with whole sperm (31.0%). In conclusion, we found that an intact membrane and tail structures of pig spermatozoa are not essential for embryo development by ICSI, and furthermore, dead porcine spermatozoa, at an early stage of necrosis caused by plasma membrane damage, support better embryo development than do live non-damaged sperm.

Ventral tail bud mesenchyme is a signaling center for tail paraxial mesoderm induction.

A large body of evidence from several systems indicates that formation of the vertebrate tail is morphogenetically continuous with gastrulation, including neural inducing activity in descendants of the gastrula organizer. However, the signaling centers and molecular events regulating tail mesoderm induction and its organized elongation remain poorly defined. In mammals, the ventral ectoderm ridge (VER) is essential to maintain ongoing formation of paraxial mesoderm and somitogenesis in cultures of intact tail. Avian tail buds contain a similar VER structure. Here, we report that the chick ventral tail bud operates as a signaling center for paraxial mesoderm induction. By using "organizer" style grafting assays to early host embryos, we found that ventral tail bud was able to induce elongated paraxial mesodermal extensions and that the ventral tail bud mesenchyme underlying the VER is both necessary and sufficient for the induction in this assay system. Our observations combined with those of others suggest that interplay between several different signaling centers in the amniote tail bud regulates the coordinate induction and elongation of axial and paraxial structures in the developing tail.

The effects of skin integrity on para-phenylenediamine hypersensitivity in BALB/c mice

Rationale Para-Phenylenediamine (PPD) is a common contact allergen found in dyes including non-permanent (henna) tattoos. In addition, several reports have demonstrated urticaria and anaphylaxis following PPD exposure. These studies were conducted to evaluate whether skin integrity modulates the Th1 versus Th2 response to PPD in murine models of hypersensitivity. Methods For the local lymph node assay (LLNA), female BALB/c mice were dosed for 3 days with vehicle (acetone) or increasing concentrations of PPD (0.05-1.0%) on the dorsal ear pinnae. Following injection of [3H]-thymidine, draining lymph nodes were radioassayed on day 6. In a phenotypic analysis assay, mice were similarly exposed to 0.05 − 6.0% PPD through intact or abraded skin for 4 days. On day 10 post-exposure, lymph nodes were excised to evaluate B220+ and IgE+B220+ cell populations. In a 3-week time course study, mice were exposed to PPD through intact or abraded skin and tail bled for serum IgE analysis at 2 and 3 weeks post initial exposure. Results A significant response was observed in the LLNA and phenotypic analysis assays at concentrations as low as 0.5 and 2.0%, respectively. Following 3 weeks of PPD exposure, IgE levels reached statistical significance ( p<0.05) in the high dose abraded skin group, however only a modest increase in IgE was observed. Conclusions These studies revealed that PPD is a potent contact sensitizer in BALB/c mice while demonstrating only weak IgE inducing potential even when exposure is through abraded skin.

IL-12 p40 homodimer-dependent macrophage chemotaxis and respiratory viral inflammation are mediated through IL-12 receptor beta 1.

Leukocyte recruitment to the airway lumen is a central feature of inflammatory conditions such as asthma and respiratory viral infection. Characterization of mediators that regulate leukocyte recruitment in these conditions revealed increased IL-12 p40 homodimer (p80) levels were associated with enhanced airway macrophage accumulation. To examine this association, we used in vivo and in vitro assays to demonstrate p80, but not IL-12 or p40, provided a macrophage chemoattractant signal. Macrophages from genetically deficient mice indicated p80-dependent chemotaxis was independent of IL-12 and required IL-12Rbeta1 (Rbeta1) expression. Furthermore, analysis of murine cell lines and primary culture macrophages revealed Rbeta1 expression, with an intact cytoplasmic tail, was necessary and sufficient to mediate p80-dependent chemotaxis. To examine the role for Rbeta1 in mediating macrophage accumulation in vivo, we contrasted Sendai virus-driven airway inflammation in wild-type and Rbeta1-deficient mice. Despite similar viral burden and production of the macrophage chemoattractant p80, the Rbeta1-deficient mice displayed a selective decrease in airway macrophage accumulation and resistance to viral-dependent mortality. Thus, Rbeta1 mediates p80-dependent macrophage chemotaxis and inhibition of the p80-Rbeta1 interaction may provide a novel anti-inflammatory strategy to manipulate the inflammation associated with asthma and respiratory viral infection.

Carboxyl-terminal truncations of human anion exchanger impair its trafficking to the plasma membrane.

The human anion exchanger AE1 (Band 3) is an abundant glycoprotein localized in plasma membrane of red cells and is responsible for the electro-neutral exchange of chloride for bicarbonate. In order to determine the role of the carboxyl-terminal tail of AE1 in its expression, function and trafficking to the plasma membrane, we generated a series of five constructs encoding truncation mutants missing the last 5 (Delta5), 11 (Delta11), 15 (Delta15), 20 (Delta20) or 35 (Delta35) amino-acids. In transiently transfected HEK 293 cells, immunoblotting of whole cell extracts showed that all the proteins were expressed at the same level as full-length AE1, except Delta20 and particularly Delta35, which showed a reduced expression. Furthermore, the last 15 amino-acids were not required for AE1 folding in the membrane, since Delta5, Delta11 and Delta15 were able to bind to an inhibitor affinity matrix, while Delta20 and Delta35 exhibited poor binding. Immunofluorescence and deglycosylation results showed that Delta15 and Delta11 were retained intracellularly, whereas a lower amount of Delta5 compared with WT trafficked to the plasma membrane. These results indicate that an intact C-terminal tail of human AE1 is important for efficient AE1 trafficking to the plasma membrane.

The role of segment 32-47 of cholecystokinin receptor type A in CCK8 binding: synthesis, nuclear magnetic resonance, circular dichroism and fluorescence studies.

The segment 32-47 of the N-terminal extracellular domain of the type A cholecystokinn receptor, CCK(A)-R(32-47), was synthesized and structurally characterized in a membrane mimicking environment by CD, NMR and molecular dynamics calculations. The region of CCK(A)-R(32-47) encompassing residues 39-46 adopted a well-defined secondary structure in the presence of DPC micelles, whereas the conformation of the N-terminal region (segment 32-37) could not be uniquely defined by the NOE derived distance constraints because of local flexibility. The conformation of the binding domain of CCK(A)-R(32-47) was different from that found for the Intact N-terminal receptor tail, CCK(A)-R(1-47). To assess whether CCK(A)-R(32-47) was still able to bind the nonsulfated cholecystokinin C-terminal octapeptide, CCK8, a series of titrations was carried out in SDS and DPC micelles, and the binding interaction was followed by fluorescence spectroscopy. These titrations gave no evidence for complex formation, whereas a high binding affinity was found between CCK(A)-R(1-47) and CCK8. The different affinities for the ligand shown by CCK(A)-R(32-47) and CCK(A)-R(1-47) were paralleled by different interaction modes between the receptor segments and the micelles.The interaction of CCK(A)-R(32-47) with DPC micelles was much weaker than that of CCK(A)-R(1-47), because the former receptor segment lacks proper stabilizing contacts with the micelle surface. In the case of SDS micelles CCK(A)-R(32-47] was found to form non-micellar adducts with the detergent that prevented the onset of a functionally significant Interaction between the receptor segment and the micelle. It is concluded that tertiary structure interactions brought about by the 1-31 segment play a key role in the stabilization of the membrane bound, biologically active conformation of the N-terminal extracellular tail of the CCKA receptor.

Molecular Mechanisms of Pre-T Cell Receptor-induced Survival

En route to maturing as T cell receptor (TCR) alphabeta-expressing cells, the development of thymocytes is contingent on expression of a pre-TCR complex comprising a TCRbeta chain paired with a surrogate TCRalpha chain, pre-Talpha (pTalpha). The pre-TCR has been proposed to promote cell survival, proliferation, differentiation, and lineage commitment. However, the precise molecular mechanisms governing this variety of effects remain elusive. Here, we present a cellular system designed to biochemically dissect signals elicited upon pre-TCR expression. Using the T cell line 4G4 stably transfected with one of the two known pTalpha isoforms or selective pTalpha deletion mutants and TCRbeta, we were able to observe that expression of a functional pre-TCR complex is sufficient to control the levels of surface Fas protein, the stimulation of mitogen-activated and stress-regulated kinases, and the activation status of the p53 antioncogene. We demonstrate that this regulation has a major impact on the expression of important regulators of apoptosis, such as Bcl-2 family members, and the cell cycle, such as p21(WAF). Furthermore, we show here that cells expressing a functional pre-TCR are more resistant to different types of DNA damage-induced apoptosis and that these effects are contingent on an intact cytoplasmic tail of pTalpha. We finally propose that the presence of a functional pre-TCR complex triggers many intracellular pathways capable of driving and ensuring thymocyte survival in the presence of DNA damage.

Mechanism of Interaction between Leucine-based Sorting Signals from the Invariant Chain and Clathrin-associated Adaptor Protein Complexes AP1 and AP2

The cytoplasmic tail of the invariant chain contains two leucine-based sorting signals, and each of those seems sufficient to route the invariant chain to its intracellular destination in either normal or polarized cells. It is believed that the intracellular routing of the invariant chain is mediated by its interactions with the clathrin-associated adaptor protein complexes AP1 and AP2. We () have previously demonstrated the in vitro interactions between the cytoplasmic tail of the invariant chain and AP1/AP2 complexes. These interactions were specific and depended on the critical leucine residues in the invariant chain's sorting signals. In the present study, we decided to investigate the molecular mechanism of these interactions. To this end, we constructed a set of glutathione S-transferase fusion proteins that contained the intact cytoplasmic tail of the invariant chain and its various mutants to define residues important for its interactions with AP1 and AP-2. Our results demonstrated the importance of several residues other than the critical leucine residues for such interactions. A strong correlation between in vitro binding of AP2 to the invariant chain and in vivo internalization of the invariant chain was observed, confirming the primary role of AP2 in recognition of endocytic signals. In addition, we demonstrated different requirements for AP1 and AP2 binding to cytoplasmic tail of the invariant chain, which may reflect that the different sorting pathways mediated by AP1 and AP2 involve their recognition of the primary structure of the sorting signal.

Distinct Domains of αIIbβ3 Support Different Aspects of Outside-In Signal Transduction and Platelet Activation Induced by LSARLAF, an αIIbβ3 Interacting Peptide

The peptide LSARLAF causes alphaIIbeta3-dependent platelet activation exemplified by secretion, aggregation, spreading and adhesion on fibrinogen, and tyrosine phosphorylation. alphaIIIbeta3-dependent outside-in signal transduction induced by LSARLAF was investigated in variant thrombasthenic platelets which lack most of the cytoplasmic domain of the integrin beta3 subunit (alphaIIbbeta3 delta724). These studies revealed that only certain aspects of this alphaIIbbeta3-dependent outside-in signaling were affected by the beta3 truncation. Specifically, alphaIIbbeta3 delta724 supported LSARLAF-induced platelet aggregation, agglutination and secretion, but failed to trigger cytoskeletal reorganization and platelet spreading on fibrinogen, despite the fact that PMA-induced non alphaIIbbeta3 mediated signaling caused spreading of these platelets on fibrinogen. Thus, distinct domains of alphaIIbbeta3 are required to support different aspects of LSARLAF-induced platelet activation. Furthermore, these studies suggest that not all alphaIIbbeta3-dependent platelet responses require an intact beta3 cytoplasmic tail.

Wound Currents Following Amputation of Tail Tip in the Japanese Newt, Cynops pyrrhogaster

Abstract When tail tips of the Japanese newt (Cynops pyrrhogaster pyrrhogaster) were amputated, large ionic currents were detected around the stump using a vibrating probe system. The initial wound currents flowed outwardly through the whole stump, and then localized within 24 hr toward the central region of the stump. Current densities reached a peak of about 450 μA • cm−2 (about 900 times greater than before amputation) within one half of a day after amputation, and returned to levels similar to the intact tail tip by 4 days, i.e., the wound currents were transient. The swelling of the stump end (regenerate or blastema) occurred about 7 days after amputation. The major ion carrying the currents is thought to be Na+, because the currents were strongly inhibited by 0.5 mM ouabain added in the external medium or by Na+-deficient medium. These findings are consistent with those of newt limb stump.

Pertussis toxin-sensitive G(i)-proteins and intracellular calcium sensitivity of vasoconstriction in the intact rat tail artery.

1. We studied the involvement of pertussis toxin (PTX)-sensitive G-proteins in the sensitivity of arterial constriction to intracellular calcium ([Ca(2+)](i)) mobilization. 2. Vasoconstriction was measured in vitro in perfused, de-endothelialized rat tail arteries loaded with the calcium-sensitive dye, fura-2 and treated or not with PTX (30 - 1000 ng ml(-1)). Arteries were stimulated with noradrenaline (NA, 0.1 - 100 microM) or KCl (15 - 120 mM). 3. KCl elicited a smaller vasoconstrictor response (E(max)=94+/-8 mmHg) than NA (E(max)=198+/-9 mmHg) although [Ca(2+)](i) mobilization was similar (E(max)=123+/-8 and 135+/-7 nM for KCl and NA, respectively). PTX (1000 ng ml(-1)) had no effect on [Ca(2+)](i) mobilization but lowered NA- (but not KCl-) induced vasoconstriction (E(max)=118+/-7 mmHg). 4. G(i/o)-proteins were revealed by immunoblotting with anti-G(i alpha) and anti-G(o alpha) antibodies in membranes prepared from de-endothelialized tail arteries. [alpha(32)P]-ADP-ribosylation of G-proteins by PTX (1000 ng ml(-1)) was demonstrated in the intact rat tail artery (pixels in the absence of PTX: 3150, presence: 25053). 5. In conclusion, we suggest that smooth muscle cells possess a PTX-sensitive G(i)-protein-mediated intracellular pathway which amplifies [Ca(2+)](i) sensitivity of contraction in the presence of agonists such as NA.

Co-induction of nitric oxide synthase, Bcl-2 and growth-associated protein-43 in spinal motoneurons during axon regeneration in the lizard tail

In lizards, tail loss transects spinal nerves and the cut axons elongate in the regrowing tail, providing a natural paradigm of robust regenerative response of injured spinal motoneurons. We previously ascertained that these events involve nitric oxide synthase induction in the axotomized motoneurons, suggesting a correlation of this enzyme with regeneration-associated gene expression. Here we investigated, in lizards, whether the cell death repressor Bcl-2 protein and growth-associated protein-43 (GAP-43) were also induced in motoneurons that innervate the regenerated tail in the first month post-caudotomy. Single and multiple immunocytochemical techniques, and quantitative image analysis, were performed. Nitric oxide synthase, GAP-43 or Bcl-2 immunoreactivity was very low or absent in spinal motoneurons of control lizards with intact tail. Nitric oxide synthase and GAP-43 were induced during the first month post-caudotomy in more than 75% of motoneurons which innnervate the regenerate. Bcl-2 was induced in approximately 95% of these motoneurons at five and 15 days, and in about 35% at one month. The intensity of Bcl-2 and GAP-43 immunostaining peaked at five days, and nitric oxide synthase at 15 days; immunoreactivity to these proteins was still significantly high at one month. Immunofluorescence revealed co-localization of nitric oxide synthase, GAP-43 and Bcl-2 in the vast majority of motoneurons at five and 15 days post-caudotomy. These findings demonstrate that co-induction of nitric oxide synthase, Bcl-2 and GAP-43 may be part of the molecular repertoire of injured motoneurons committed to survival and axon regeneration, and strongly favor a role of nitric oxide synthase in motoneuron plasticity.

Plastic changes and nitric oxide synthase induction in neurons which innervate the regenerated tail of the lizard Gekko gecko: II. The response of dorsal root ganglion cells to tail amputation and regeneration

The lizard tail regenerates after amputation, which severs the spinal cord and spinal nerves. Dorsal root ganglia (DRGs) do not regenerate in the regrowing tail, which is innervated by DRGs rostral to the amputation. With Nissl staining, NADPH-diaphorase histochemistry and nitric oxide synthase (NOS) immunohistochemistry, we investigated NOS expression and its relationship with structural changes in DRG neurons of caudotomized lizards. First, by horseradish peroxidase retrograde tracing we here provided evidence that the sensory innervation of the regenerated tail derives only from the three pairs of DRGs rostral to the amputation plane. These ganglia were then analyzed in control animals with original intact tail, at 5, 15 and 30 days after caudotomy, and at 8 months in lizards with mature regenerates. Caudotomy elicited in DRG neurons marked hypertrophy that persisted after tail regeneration. In control ganglia, most neurons were lightly NADPH-diaphorase-positive, a few were unstained or intensely stained. Tail transection elicited marked staining up-regulation, and an increase in the proportion of intensely positive neurons. The staining intensity peaked in DRG neurons at 15 days and was still significantly increased in respect to controls several months after complete tail regeneration. NOS immunoreactivity in DRGs matched the histochemical findings. NADPH-diaphorase positivity was also enhanced in the dorsal horn superficial laminae of the corresponding spinal segments. We demonstrate that transection of the lizard spinal nerves, provoked by tail loss, elicits in the axotomized primary sensory neurons marked NOS enhancement, which accompanies axon elongation in the regrowing tail and persists after the end of this process.

Dynamic Ca2+ signalling in rat arterial smooth muscle cells under the control of local renin-angiotensin system.

1. We visualized the changes in intracellular Ca2+ concentration ([Ca2+]i), using fluo-3 as an indicator, in individual smooth muscle cells within intact rat tail artery preparations. 2. On average in about 45 % of the vascular smooth muscle cells we found spontaneous Ca2+ waves and oscillations ( approximately 0.13 Hz), which we refer to here as Ca2+ ripples because the peak amplitude of [Ca2+]i was about one-seventh of that of Ca2+ oscillations evoked by noradrenaline. 3. We also found another pattern of spontaneous Ca2+ transients often in groups of two to three cells. They were rarely observed and are referred to as Ca2+ flashes because their peak amplitude was nearly twice as large as that in noradrenaline-evoked responses. 4. Sympathetic nerve activity was not considered responsible for the Ca2+ ripples, and they were abolished by inhibitors of either the Ca2+ pump in the sarcoplasmic reticulum (cyclopiazonic acid) or phospholipase C (U-73122). 5. Both angiotensin antagonists ([Sar1,Ile8]-angiotensin II and losartan) and an angiotensin converting enzyme inhibitor (captopril) inhibited the Ca2+ ripples. 6. The extracellular Ca2+-dependent tension borne by unstimulated arterial rings was reduced by the angiotensin antagonist by approximately 50 %. 7. These results indicate that the Ca2+ ripples are generated via inositol 1,4, 5-trisphosphate-induced Ca2+ release from the intracellular Ca2+ stores in response to locally produced angiotensin II, which contributes to the maintenance of vascular tone.