Transcranial low-level laser therapy enhances learning, memory, and neuroprogenitor cells after traumatic brain injury in mice.

The use of transcranial low-level laser (light) therapy (tLLLT) to treat stroke and traumatic brain injury (TBI) is attracting increasing attention. We previously showed that LLLT using an 810-nm laser 4 h after controlled cortical impact (CCI)-TBI in mice could significantly improve the neurological severity score, decrease lesion volume, and reduce Fluoro-Jade staining for degenerating neurons. We obtained some evidence for neurogenesis in the region of the lesion. We now tested the hypothesis that tLLLT can improve performance on the Morris water maze (MWM, learning, and memory) and increase neurogenesis in the hippocampus and subventricular zone (SVZ) after CCI-TBI in mice. One and (to a greater extent) three daily laser treatments commencing 4-h post-TBI improved neurological performance as measured by wire grip and motion test especially at 3 and 4 weeks post-TBI. Improvements in visible and hidden platform latency and probe tests in MWM were seen at 4 weeks. Caspase-3 expression was lower in the lesion region at 4 days post-TBI. Double-stained BrdU-NeuN (neuroprogenitor cells) was increased in the dentate gyrus and SVZ. Increases in double-cortin (DCX) and TUJ-1 were also seen. Our study results suggest that tLLLT may improve TBI both by reducing cell death in the lesion and by stimulating neurogenesis.

Hyperthermia induces cytoskeletal alterations and mitotic catastrophe in p53-deficient H1299 lung cancer cells.

Hyperthermia is used in cancer therapy, however much remains to be discovered regarding its mechanisms of action at the cellular level. In this study, the effects of hyperthermia on cell death, survival, morphology and the cytoskeleton were investigated in a non-small cell lung cancer cell line, H1299. Despite the fact that this cell line is widely used in research, it has not yet been tested for heat shock sensitivity. Cells were given a 30-min heat shock at 43.5°C and 45°C and left to recover at 37°C for 24 and 48 h. 24 h after heat shock treatment, we monitored changes in the organization of the cytoskeleton using immunofluorescence microscopy. The number of actin stress fibers was significantly reduced, microtubules formed a looser meshwork, a portion of the cells possessed multipolar mitotic spindles, whereas vimentin filaments collapsed into perinuclear complexes. 48 h following heat stress, most of the cells showed recovery of the cytoskeleton, however we observed a considerable number of giant cells that were multinucleated or contained one enlarged nucleus. The data obtained by MTT assay showed a dose-dependent decrease of cell viability, while flow cytometric analysis revealed an increase in the number of cells with externalized phosphatidylserine. The results suggest that one of the modes of heat-induced cell death in H1299 cells is mitotic catastrophe, which probably ends in apoptosis.

Whole cell microtubule analysis by flow cytometry.

Perturbation of the tubulin/microtubule dynamic in cells is perhaps the single most important mode of action of anticancer drugs. Standard methods for identifying and evaluating compounds for their ability to alter tubulin polymerization are low throughput, labor intensive, expensive, or make their assessment in vitro. Here we report a method to rapidly quantify the extent of tubulin polymerization in whole cells using flow cytometry, and we use this technique to evaluate compounds that stabilize and destabilize microtubule formation. This facile method is useful for conveniently, quantitatively, and cost-effectively comparing small molecules that perturb tubulin polymerization.

Measurement of ligand binding to tubulin by sulfhydryl reactivity.

Ligand binding can induce shifts in protein conformation. In the case of tubulin, these drug-induced confirmational changes can prevent or stabilize microtubule polymerization. 5',5'-Dithiobis(2-nitrobenzoate) (DTNB) reacts with free and accessible sulfhydryls and stoichiometrically produces a detectable product, which allows an exact measurement of reacted thiols. Since binding of small ligands may alter conformational dynamics, it may also affect the reactivity of thiols on tubulin. Differences in DTNB reactivity with thiols upon ligand binding can therefore be used to deduce binding characteristics. We will describe two methods that use tubulin cysteine reactivity with DTNB in the presence of drug to define ligand-binding characteristics.

Supporting cell characteristics in long-deafened aged mouse ears.

Significant sensory hair cell loss leads to irreversible hearing and balance deficits in humans and other mammals. Future therapeutic strategies to repair damaged mammalian auditory epithelium may involve inserting stem cells into the damaged epithelium, inducing non-sensory cells remaining in the epithelium to transdifferentiate into replacement hair cells via gene therapy, or applying growth factors. Little is currently known regarding the status and characteristics of the non-sensory cells that remain in the deafened auditory epithelium, yet this information is integral to the development of therapeutic treatments. A single high-dose injection of the aminoglycoside kanamycin coupled with a single injection of the loop diuretic furosemide was used to kill hair cells in adult mice, and the mice were examined 1 year after the drug insult. Outer hair cells are lost throughout the entire length of the cochlea and less than a third of the inner hair cells remain in the apical turn. Over 20% and 55% of apical organ of Corti support cells and spiral ganglion cells are lost, respectively. We examined the expression of several known support cell markers to investigate for possible support cell dedifferentiation in the damaged ears. The support cell markers investigated included the microtubule protein acetylated tubulin, the transcription factor Sox2, and the Notch signaling ligand Jagged1. Non-sensory epithelial cells remaining in the organ of Corti retain acetylated tubulin, Sox2 and Jagged1 expression, even when the epithelium has a monolayer-like appearance. These results suggest a lack of marked SC dedifferentiation in these aged and badly damaged ears.

[Directed differentiation of mouse embryonic stem cells into neuronal cells induced by icaritin in vitro].

OBJECTIVE: To evaluate the inductive effects of icaritin (ICT) on the directed differentiation of mouse embryonic stem (ES) cells into neuronal cells in vitro. METHODS: ES cells were cultured with embryoid body (EB) formation cultures, ICT in different concentrations was added in the cultural media and the cells were harvested in several differentiation phases. The expression spectrums of neuronal cell-specific genes and proteins were verified by semi-quantitative RT-PCR and immunocytochemistry analysis, respectively. RESULTS: Differentiation of neurocyte phenotype from ES cells was promoted by ICT in a concentration-and time-dependent manner. ICT at 10(-7)mol/L significantly enhanced the differentiation toward neuronal cells, and up to 80 % of EBs outgrowth in d 8+8 incubation. The gene expressions of beta-tubulin III in neuron and GFAP in glial cells were detected in neuronal cell phenotype derived from EBs. Furthermore, nestin was detected in precursor cells, beta-tubulin III and GFAP were detected in the generated precursor neurocytes immunocytochemically. CONCLUSION: Directed differentiation of neurons is facilitated by ICT in EB formation culture, which is associated with the expression of developmental-dependent gene and protein.

Glyphosate-induced anther indehiscence in cotton is partially temperature dependent and involves cytoskeleton and secondary wall modifications and auxin accumulation.

Yield reduction caused by late application of glyphosate to glyphosate-resistant cotton (Gossypium hirsutum; GRC) expressing CP4 5-enol-pyruvylshikmate-3-P synthase under the cauliflower mosaic virus-35S promoter has been attributed to male sterility. This study was aimed to elucidate the factors and mechanisms involved in this phenomenon. Western and tissue-print blots demonstrated a reduced expression of the transgene in anthers of GRC compared to ovules of the same plants. Glyphosate application to GRC grown at a high temperature regime after the initiation of flower buds caused a complete loss of pollen viability and inhibition of anther dehiscence, while at a moderate temperature regime only 50% of the pollen grains were disrupted and anther dehiscence was normal. Glyphosate-damaged anthers exhibited a change in the deposition of the secondary cell wall thickenings (SWT) in the endothecium cells, from the normal longitudinal orientation to a transverse orientation, and hindered septum disintegration. These changes occurred only at the high temperature regime. The reorientation of SWT in GRC was accompanied by a similar change in microtubule orientation. A similar reorientation of microtubules was also observed in Arabidopsis (Arabidopsis thaliana) seedlings expressing green fluorescent protein tubulin (tubulin alpha 6) following glyphosate treatment. Glyphosate treatment induced the accumulation of high levels of indole-3-acetic acid in GRC anthers. Cotton plants treated with 2,4-dichlorophenoxyacetic acid had male sterile flowers, with SWT abnormalities in the endothecium layer similar to those observed in glyphosate-treated plants. Our data demonstrate that glyphosate inhibits anther dehiscence by inducing changes in the microtubule and cell wall organization in the endothecium cells, which are mediated by auxin.

Exploiting the genetic diversity of Beauveria bassiana for improving the biological control of the coffee berry borer through the use of strain mixtures.

Beauveria bassiana is an entomopathogen widely used to control the coffee berry borer in Colombia, as part of an Integrated Pest Management strategy. Traditionally, the development of fungal insect pathogens as biocontrol agents in crop pests has been oriented towards the selection and formulation of elite clonal strains. Instead, we explored the potential application of genetic diversity in B. bassiana by determining the effect of strain mixtures on coffee berry borer mortality compared to clonal isolates. Genomic DNA from 11 strains was characterized using internal transcribed spacers and beta-tubulin sequences as well as amplified fragment length polymorphism markers. Cluster analysis produced three genetic groups and confirmed the low but significant intraspecific genetic diversity present among the strains. Single strain virulence towards the coffee berry borer under laboratory conditions, using 1x10(6) conidia ml(-1), ranged between 89.9 and 57.5%. All the inoculations with mixtures resulted in coinfection events. Combinations of genetically similar strains showed no significant differences when their virulences were compared. However, mixtures of genetically different strains led to both antagonism and synergism. The lowest virulence percentage (57%) was obtained by putting together the most virulent strain of each group, contrary to the highest virulence percentage (93%) that resulted from mixing the three least virulent strains. The results indicate the promising potential of designing strain mixtures as an alternative for the biocontrol of Hypothenemus hampei and other pests and provide tools for the understanding of the ecological dynamics of entomopathogen populations under natural conditions.

stathmin, a gene enriched in the amygdala, controls both learned and innate fear.

Little is known about the molecular mechanisms of learned and innate fear. We have identified stathmin, an inhibitor of microtubule formation, as highly expressed in the lateral nucleus (LA) of the amygdala as well as in the thalamic and cortical structures that send information to the LA about the conditioned (learned fear) and unconditioned stimuli (innate fear). Whole-cell recordings from amygdala slices that are isolated from stathmin knockout mice show deficits in spike-timing-dependent long-term potentiation (LTP). The knockout mice also exhibit decreased memory in amygdala-dependent fear conditioning and fail to recognize danger in innately aversive environments. By contrast, these mice do not show deficits in the water maze, a spatial task dependent on the hippocampus, where stathmin is not normally expressed. We therefore conclude that stathmin is required for the induction of LTP in afferent inputs to the amygdala and is essential in regulating both innate and learned fear.

[Contractile proteins in chemical signal transduction in plant microspores].

Involvement of contractile components in chemical signal transduction from the cell surface to the organelles was studied using unicellular systems. Neurotransmitters dopamine and serotonin as well as active forms of oxygen hydrogen peroxide and tert-butyl peroxide were used as chemical signals. Experiments were carried out on vegetative microspores of field horsetail Equisetum arvense and generative microspores (pollen) of amaryllis Hippeastrum hybridum treated with cytochalasin B (an inhibitor of actin polymerization in microfilaments), colchicine, and vinblastine (inhibitors of tubulin polymerization in microtubules). Both types of thus treated microspores demonstrated suppressed development, particularly, for cytochalasin B treatment. At the same time, an increased typical blue fluorescence of certain cell regions (along the cell wall and around nuclei and chloroplasts) where the corresponding contractile proteins could reside was observed. In contrast to anticontractile agents, dopamine, serotonin B, and the peroxides stimulated microspore germination. Microspore pretreatment with cytochalasin B and colchicine followed by the treatment with serotonin, dopamine, or the peroxides decreased the germination rate. Involvement of actin and tubulin in chemical signal transduction from the cell surface to the nucleus is proposed.

Movement ability of rye terminal neocentromeres.

Rye terminal neocentromeres were analyzed in various aspects. Plants with and without neocentromeres were crossed to determine the possible genetic control on their formation. The segregation obtained in our work is consistent with the hypothesis of two trans-acting genes determining neocentric activity in such a way that individuals with no neocentromeres at all would carry all non-activating alleles, whereas one activating allele might permit the activation of a few neocentromeres. Individuals with four activating alleles would show the maximum frequency of neocentromeres per cell. Anti-tubulin immunolabelling was used to visualize the interaction between the neocentromeres and the microtubules. In most cases an end-on interaction between neocentromeres and microtubules was observed, but a few neocentromeres were observed free of them. Spikes were irradiated at early meiosis to determine whether acentric fragments carrying subtelomeric heterochromatin were able to behave as neocentromeres. In no case were acentric fragments observed to form an extension polewards as they did in whole chromosomes. Broken chromosomes joined by a thin thread of chromatin to the centromeric region

Pseudoneoplastic proliferation of histiocytes with paclitaxel-induced ultrastructural changes in a mastectomy specimen.

A 49-year-old Hispanic woman with a T4N1M0 infiltrating duct carcinoma of the left breast underwent four courses of FAC (doxorubicin 86 mg, 5-fluorouracil 860 mg, cyclophosphamide 86 mg, and dexamethasone 10 mg) adjuvant chemotherapy plus four courses of paclitaxel (Taxol; Bristol-Myers Squibb Oncology, Princeton, NJ) and subsequent mastectomy. The tumor shrunk from 6.5 cm to 2.5 cm after the treatment. The residual tumor in the surgical specimen measured 1.5 cm with eight positive out of 24 axillary lymph nodes. The tumor showed typical chemotherapy changes and a massive proliferation of histiocytes that mimicked a neoplasm. A nodular proliferation of the same cells in one axillary node raised the impression of a second malignant tumor in the breast spreading to the node. The histiocytic cells contained lamellar and coarse periodic acid-Schiff-positive material distending their cytoplasm and they were strongly positive for CD68 and negative for CD1a, pan keratin, and S-100. These findings ruled out histiocytoid carcinoma, granular cell tumor, and Erdheim-Chester disease. The proliferating histiocytes had ultrastructural findings of paclitaxel-induced cytotoxicity with disorganized stacks of intermediate filaments positive for vimentin by immunostains and fewer masses of tubulin. The treated breast carcinoma cells were tubulin-positive but the proliferating histiocytes were tubulin-negative.

[Reorganization of the tubulin and actin cytoskeleton under acclimation and abscisic acid treatment of Triticum aestivum L. plants]. / Reorganizatsiia tubulinovogo i aktinovogo tsitoskeleta pri zakalivanii rastenii Triticum aestivum L. k kholodu i deistvii abstsizovoi kisloty.

Only scanty and contradictory data are available concerning effects of low temperatures and ABA on the structural organization of microtubules (MTs) and microfilaments (MFs), and no information exists on the interaction of these parameters at cold acclimation of plants. Therefore, in cold acclimate and ABA-treated winter wheat plants, a comparative study was made of the state (localization, orientation, structure) and stability of actin and tubulin cytoskeleton in root cells taken from different zones, using indirect immunofluorescent microscope. The plant cold acclimation caused MT aggregation, the rise of MT and MF fluorescence, and the increase of their stability (a decrease of oryzalin effect) mainly in the root differentiation zone, that may testify to the strengthening of contacts between MTs and MFs. Like the cold acclimation, ABA induced the formation of MT bunches only in meristem and elongation zone cells. However in the zone of differentiation, the hormone stimulated the increase of tubulin structure stability, well correlating with a decrease in MT content, aggregation degree, and immunofluorescence, and, in addition with a complete depolymerization of MFs. Low temperatures removed the hormone effect on the structural organization of tubulin and actin cytoskeleton in the zone of differentiation. It is suggested that MT destruction, the decrease of instable MT populations, and the increase of stable MT populations may slow down growth processes in ABA-treated plants, similarly as in seedlings being on the initial stages of cold acclimation. By the end of this process, the induction of plant growth is determined evidently by the increase in the number of instable, highly labile MT populations, and in the status of MF polymerization.

Characterization of mitotic neurons derived from adult rat hypothalamus and brain stem.

Embryonic or neonatal rat neurons retain plasticity and are readily grown in tissue culture, but neurons of the adult brain were thought to be terminally differentiated and therefore difficult to culture. Recent studies, however, suggest that it may be possible to culture differentiated neurons from the hippocampus of adult rats. We modified these procedures to grow differentiated neurons from adult rat hypothalamus and brain stem. At day 7 in tissue culture and beyond, the predominant cell types in hypothalamic and brain stem cultures had a stellate morphology and could be subdivided into two distinct groups, one of which stained with antibodies to the immature neuron marker alpha-internexin, while the other stained with the astrocyte marker GFAP. The alpha-internexin positive cells were mitotic and grew to form a characteristic two-dimensional cellular network. These alpha-internexin positive cells coimmunostained for the neuronal markers MAP2, type III beta-tubulin, and tau, and also bound tetanus toxin, but were negative for the oligodendrocyte marker GalC and also for the neurofilament triplet proteins NF-L, NF-M, and NF-H, markers of more mature neurons. Patch-clamp analysis of these alpha-internexin positive cells revealed small Ca(2+) currents with a peak current of -0.5 +/- 0.1 pA/pF at a membrane potential of -20 mV (n = 5) and half-maximal activation at -30 mV (n = 5). Na(+) currents with a peak current density of -154.5 +/- 49.8 pA/pF at a membrane potential of -15 mV (n = 5) were also present. We also show that these cells can be frozen and regrown in tissue culture and that they can be efficiently infected by viral vectors. These cells therefore have the immunological and electrophysiological properties of immature mitotic neurons and should be useful in a variety of future studies of neuronal differentiation and function.

Molecular cloning and characterization of a metal responsive Chironomus tentans alpha-tubulin cDNA.

Metal pollution of aquatic ecosystems is a problem of economic and health importance. Sensitive molecular biomarkers of metal exposure are sorely needed. We have isolated a cDNA from the midge Chironomus tentans that is transcribed in all organs and developmental stages. The cDNA encodes a protein, designated Chironomus tentans alpha-tubulin 1 (CTTUB1), which has significant similarities with invertebrate and vertebrate alpha-tubulins. CTTUB1 is abundantly transcribed in embryos and to a lesser extent in adults and larvae. CTTUB1 RNA and protein abundances are increased in larvae exposed to copper or cadmium. The pattern of cellular distribution of CTTUB1 protein in the midgut epithelial cells was radically affected by cadmium. In the midgut cells of unexposed larvae, CTTUB1 was found evenly distributed throughout the cytoplasm, while in cadmium-exposed larvae, CTTUB1 was mostly concentrated along the basolateral plasma membrane. A mechanism for the regulation of alpha-tubulin synthesis by cadmium is proposed. This is the first report on the isolation of a metal responsive gene from a neartic aquatic insect.

Structure-activity relationship of polyisoprenyl benzophenones from Garcinia pyrifera on the tubulin/microtubule system.

Microtubule disassembly inhibitory properties have been established for the known polyisoprenylated benzophenones xanthochymol (1a) and guttiferone E (1b). The compounds were isolated from the fruits of Garcinia pyrifera collected in Malaysia. A structure-activity relationship study, including natural and semisynthetic derivatives, delineated some structural features necessary for the interaction with tubulin within this compound class.

Immunocytochemical localization of actin and tubulin in the integument of land crab (Gecarcinus lateralis) and lobster (Homarus americanus).

The crustacean integument consists of the exoskeleton and underlying epithelium and associated tissues. The epithelium, which is composed of a single layer of cells, is responsible for the cyclical breakdown and synthesis of the exoskeleton associated with molting (ecdysis). During premolt (proecdysis) the epithelial cells lengthen and secrete the two outermost layers (epicuticle and exocuticle) of the new exoskeleton while partially degrading the two innermost layers (endocuticle and membranous layer) of the overlying old exoskeleton. This increased cellular activity is associated with increased protein synthesis and a change in cell shape from cuboidal to columnar. The cytoskeleton, composed of microfilaments (actin) and microtubules (tubulin), plays important roles in the intracellular organization and motility of eukaryotic cells. Immunoblot analysis shows that the land crab exoskeleton contains actin, tubulin, and actin-related proteins (Varadaraj et al. 1996. Gene 171:177-184). In the present study, immunocytochemistry of land crab and lobster integument showed that both proteins were localized in various cell types, including epithelia, connective tissue, tendinal cells, and blood vessels. Muscle immunostained for actin and myosin, but not for tubulin. The membranous layer of land crab (the other layers of the exoskeleton were not examined) and membranous layer and endocuticle of lobster also reacted specifically with anti-beta-actin and anti-alpha-tubulin monoclonal antibodies, but not with an anti-myosin heavy chain antibody. During proecdysis immunolabeling of the membranous layer decreased probably due to protein degradation. The staining intensity for actin and tubulin in the proecdysial epithelium was similar to that in the intermolt (anecdysial) epithelium, suggesting that there was a net accumulation of both proteins proportional to the increase in cellular volume. These results support the previous biochemical analyses and, more specifically, localize actin and tubulin in exoskeletal structures, suggesting that they may serve both intracellular and extracellular functions in crustaceans. J. Exp. Zool. 286:329-342, 2000.

A new look at embryonic development of the visual system in decapod crustaceans: neuropil formation, neurogenesis, and apoptotic cell death.

In recent years, comparing the structure and development of the central nervous system in crustaceans has provided new insights into the phylogenetic relationships of arthropods. Furthermore, the structural evolution of the compound eyes and optic ganglia of adult arthropods has been discussed, but it was not possible to compare the ontogeny of arthropod visual systems, owing to the lack of data on species other than insects. In the present report, we studied the development of the crustacean visual system by examining neurogenesis, neuropil formation, and apoptotic cell death in embryos of the American lobster, Homarus americanus, the spider crab, Hyas araneus, and the caridean shrimp, Palaemonetes argentinus, and compare these processes with those found in insects. Our results on the patterns of stem cell proliferation provide evidence that in decapod crustaceans and hemimetabolous insects, there exist considerable similarities in the mechanisms by which accretion of the compound eyes and growth of the optic lobes is achieved, suggesting an evolutionary conservation of these mechanisms.

Confocal analysis of primary cilia structure and colocalization with the Golgi apparatus in chondrocytes and aortic smooth muscle cells.

Detyrosinated and acetylated alpha-tubulins represent a stable pool of tubulin typically associated with microtubules of the centrosome and primary cilium of eukaryotic cells. Although primary cilium-centrosome and centrosome-Golgi relationships have been identified independently, the precise structural relationship between the primary cilium and Golgi has yet to be specifically defined. Confocal immunohistochemistry was used to localize detyrosinated (ID5) and acetylated (6-11B-1) tubulin antibodies in primary cilia of chondrocytes and smooth muscle cells, and to demonstrate their relationship to the Golgi complex identified by complementary lectin staining with wheat germ agglutinin. The results demonstrate the distribution and inherent structural variation of primary cilia tubulins, and the anatomical interrelationship between the primary cilium, the Golgi apparatus and the nucleus. We suggest that these interrelationships may form part of a functional feedback mechanism which could facilitate the directed secretion of newly synthesized connective tissue macromolecules.

Embryonic taste buds develop in the absence of innervation.

It has been hypothesized that taste buds are induced by contact with developing cranial nerve fibers late in embryonic development, since descriptive studies indicate that during embryonic development taste cell differentiation occurs concomitantly with or slightly following the advent of innervation. However, experimental evidence delineating the role of innervation in taste bud development is sparse and equivocal. Using two complementary experimental approaches, we demonstrate that taste cells differentiate fully in the complete absence of innervation. When the presumptive oropharyngeal region was taken from a donor axolotl embryo, prior to its innervation and development of taste buds, and grafted ectopically on to the trunk of a host embryo, the graft developed well-differentiated taste buds. Although grafts were invaded by branches of local spinal nerves, these neurites were rarely found near ectopic taste cells. When the oropharyngeal region was raised in culture, numerous taste buds were generated in the complete absence of neural elements. Taste buds in grafts and in explants were identical to those found in situ both in terms of their morphology and their expression of calretinin and serotonin immunoreactivity. Our findings indicate that innervation is not necessary for complete differentiation of taste receptor cells. We propose that taste buds are either induced in response to signals from other tissues, such as the neural crest, or arise independently through intrinsic patterning of the local epithelium.