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Bilateral organization of unilaterally generated activity in lumbar spinal motoneurons of the rat.

The spinal nucleus of the bulbocavernosus (SNB) is a medially located, bilaterally organized sexually dimorphic motor nucleus in the lumbar spinal cord of the male rat. To begin to assess the potential functional significance of this bilateral organization, we recorded ipsi- and contralateral SNB motor nerve activity following unilateral spinal stimulation and examined the timing, pattern, and recruitment of population motoneuron activity. A possible mechanism for bilateral communication, gap junctional intercellular communication, was also investigated because dye coupling experiments indicate an extensive syncytium in which SNB motoneurons are coupled with each other and neighboring interneurons. An in vivo peripheral nerve recording paradigm was used: a bipolar stimulating electrode was placed on dorsal root L6, and bipolar recording electrodes were placed bilaterally on the SNB motor nerves. All processes were severed distal to electrode placement to isolate the central preparation; recruitment curves of motoneuronal activity were then generated. Amplitude of peak to peak recruitment was greater in the contralateral motor nerve than in the ipsilateral nerve. Response latency, Fourier transform and spike counts showed no evidence of ipsi/contralateral asymmetry. Recruitment was attenuated both ipsi- and contralaterally after pharmacological gap junction blockade, but antidromic stimulation could not drive activity in contralateral motor axons. These results indicate that unilateral input to the SNB may be differentially modulated to produce functionally distinct output in the two separate halves of the nucleus. We also discuss the potential modulatory role of gap junctions in the activity of the SNB.

Afferent Pathways↗

Functional associations among trauma, PTSD, and substance-related disorders.

This review article presents several potential functional pathways which may explain the frequent co-occurrence of PTSD and substance abuse disorders in traumatized individuals. Emerging empirical studies which have examined these potential pathways are reviewed, including studies on relative order of onset, PTSD patients' perceptions of various drug effects, comparisons of PTSD patients with and without comorbid substance use disorders, and correlational studies examining the relations between severity of specific PTSD symptom clusters and substance disorder symptoms. Research on the acute and chronic effects of alcohol and other drugs on cognitive and physiological variables relevant to PTSD intrusion and arousal symptoms is reviewed to highlight ways in which these two sets of PTSD symptoms might be functionally interrelated with substance abuse. Finally, based on these findings, recommendations are made for the treatment of individuals with comorbid PTSD-substance use disorders.

Affective Symptoms↗

Functional dissection of a eukaryotic dicistronic gene: transgenic stonedB, but not stonedA, restores normal synaptic properties to Drosophila stoned mutants.

The dicistronic Drosophila stoned mRNA produces two proteins, stonedA and stonedB, that are localized at nerve terminals. While the stoned locus is required for synaptic-vesicle cycling in neurons, distinct or overlapping synaptic functions of stonedA and stonedB have not been clearly identified. Potential functions of stoned products in nonneuronal cells remain entirely unexplored in vivo. Transgene-based analyses presented here demonstrate that exclusively neuronal expression of a dicistronic stoned cDNA is sufficient for rescue of defects observed in lethal and viable stoned mutants. Significantly, expression of a monocistronic stonedB trangene is sufficient for rescuing various phenotypic deficits of stoned mutants, including those in organismal viability, evoked transmitter release, and synaptotagmin retrieval from the plasma membrane. In contrast, a stonedA transgene does not alleviate any stoned mutant phenotype. Novel phenotypic analyses demonstrate that, in addition to regulation of presynaptic function, stoned is required for regulating normal growth and morphology of the motor terminal; however, this developmental function is also provided by a stonedB transgene. Our data, although most consistent with a hypothesis in which stonedA is a dispensable protein, are limited by the absence of a true null allele for stoned due to partial restoration of presynaptic stonedA by transgenically provided stonedB. Careful analysis of the effects of the monocistronic transgenes together and in isolation clearly reveals that the presence of presynaptic stonedA is dependent on stonedB. Together, our findings improve understanding of the functional relationship between stonedA and stonedB and elaborate significantly on the in vivo functions of stonins, recently discovered phylogenetically conserved stonedB homologs that represent a new family of "orphan" medium (mu) chains of adaptor complexes involved in vesicle formation. Data presented here also provide new insight into potential mechanisms that underlie translation and evolution of the dicistronic stoned mRNA.

Animals↗

Human CD1d functions as a transplantation antigen and a restriction element in mice.

To study the potential functions of human CD1d (hCD1d), we developed transgenic (Tg) mice that ectopically express hCD1d under the control of H-2K(b) promoter. High levels of hCD1d expression were detected in all Tg tissues tested. Skin grafts from the K(b)/hCD1d Tg mice were rapidly rejected by MHC-matched non-Tg recipient mice, suggesting that hCD1d can act as transplantation Ags. Furthermore, we were able to elicit hCD1d-restricted CD8(+) CTLs from mice immunized with K(b)/hCD1d Tg splenocytes. These CTLs express TCR rearrangements that are distinct from invariant TCR of NK T cells, and secrete significant amounts of IFN-gamma upon Ag stimulation. Analysis with various hCD1d-expressing targets and use of Ag presentation inhibitors indicated the recognition of hCD1d by CTLs did not involve species or tissue-specific ligands nor require the processing pathways of endosomes or proteasomes. Additionally, the reactivity of hCD1d-specific CTLs was not affected by acid stripping followed by brefeldin A treatment, suggesting that CTLs may recognize a ligand/hCD1d complex that is resistant to acid denaturation, or empty hCD1d molecules. Our results show that hCD1d can function as an alloantigen for CD8(+) CTLs. The hCD1d Tg mice provide a versatile model for the study of hCD1d-restricted cytolytic responses to microbial Ags.

Animals↗

A method for real-time processing to study recovery functions of evoked potentials.

A new method for evaluating the recovery function of evoked potential (EP) in real time was reported. To record the test response of paired stimulation, the responses to alternate presentation of unpaired stimulus and several kinds of paired stimuli with various interstimulus intervals were averaged respectively and subtraction was made in real time. The EP's to the test stimulus obtained by the proposed method were proved to be accurate even under the changing recording conditions. A formula for computing the recovery correlation factor was derived as a measure of the recovery function of the recorded EP, and the recovery curves for three subjects were obtained.

Adult↗

Interplay between DNA and RNA methylation shapes cancer cell plasticity.

Cellular plasticity refers to the ability of healthy cells to shift between phenotypic states and modify their characteristics to maintain tissue homeostasis and integrity. In the tumor context, cancer stem cells (CSCs) exploit this flexibility to withstand stress, facilitate tumor dissemination, and evade therapeutic interventions. Epigenetic regulation, particularly DNA methylation at CpG sites, is recognized as a well-known driver of tumor plasticity by repressing differentiation programs through modulation of chromatin accessibility. More recently, RNA modifications (epitranscriptomics) have emerged as crucial post-transcriptional regulators of gene expression that shape RNA fate and function. Among these, N6-methyladenosine (m6A), 5-methylcytosine (m5C), N1-methyladenosine (m1A), and N7-methylguanosine (m7G) contribute to the regulation of cell identity by modulating stemness-differentiation balance, stress adaptation, and epithelial-to-mesenchymal transition (EMT). Notably, dysregulation of both DNA and RNA methylation signatures is frequently observed in tumors, suggesting potential functional interactions between these regulatory layers. Emerging evidence indicates that DNA CpG methylation and RNA methylation pathways may cooperate to influence stemness, survival, and EMT-associated signaling, thereby supporting CSCs' plasticity. Although the molecular mechanisms underlying this crosstalk remain incompletely understood, accumulating studies suggest that DNA and RNA methylation could converge within interconnected regulatory networks that contribute to the control of cancer cell identity. A deeper understanding of these interactions may uncover novel vulnerabilities for targeting tumor plasticity. In this review, we summarize the current knowledge on the interplay between DNA and RNA methylation in regulating tumor plasticity, highlighting emerging mechanistic insights, functional interactions, and potential implications for future epigenetic and epitranscriptomic therapeutic strategies.

Humans↗

Functional interaction between CFTR and Cx45 gap junction channels expressed in oocytes.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride (Cl(-)) channel known to influence the function of other channels, including connexin channels. To further study potential functional interactions between CFTR and gap junction channels, we have co-expressed CFTR and connexin45 (Cx45) in Xenopus oocytes and monitored junctional conductance and voltage sensitivity by dual voltage clamp electrophysiology. In single oocytes expressing CFTR, an increase in cAMP caused by forskolin application induced a Cl(-) current and increased membrane conductance; application of diphenylamine carboxylic acid (CFTR blocker) readily blocked the Cl(-) current. With co-expression of CFTR and Cx45, application of forskolin to paired oocytes induced a typical outward current and increased junctional conductance (G(j)). In addition, the presence of CFTR reduced the transjunctional voltage sensitivity of Cx45 channels without affecting the kinetics of junctional current inactivation. The drop in voltage sensitivity was further enhanced by forskolin application. The data indicate that CFTR influences cell-to-cell coupling mediated by Cx45 channels.

Animals↗

Pattern electroretinogram, visual evoked potential and psychophysical functions in maculopathy.

To compare pattern electroretinograms and visual evoked potentials with psychophysical examinations, such as visual acuity, static (automated) perimetry and color vision in unilateral maculopathies of various origins, 20 patients with unilateral retinal diseases within the macula and the posterior pole were tested. Pattern electroretinography, visual evoked potential testing and static perimetry (Octopus program M1) were performed with three different test field sizes (20 degrees x 20 degrees, 10 degrees x 10 degrees and 6 degrees x 6 degrees). The best correlation in all three test field sizes was found between visual acuity, static perimetry and visual evoked potential. This result is surprising, since central area defined functions (visual evoked potentials, visual acuity) correlated well with a total area integrating function (mean defect in static perimetry. The pattern electroretinogram, which seems to reflect an area-related function as well, showed a correlation to static perimetry only in the smaller 10 degrees x 10 degrees and 6 degrees x 6 degrees fields and not a significant correlation in the 20 degrees x 20 degrees field. Smaller stimulation fields may therefore produce sharper results in pattern electroretinographic testing. There was no correlation between pattern electroretinograms and visual evoked potentials or visual acuity. The pattern electroretinogram was recorded under monocular and binocular viewing conditions. In 60% of the patients, the amplitude of the affected eye was more reduced in the monocular than the binocular viewing condition; the healthy fellow eye controlled stable fixation of the affected eye more readily during binocular pattern electroretinogram registration. The degree of the color vision disturbance (C-index, desaturated panel D-15 test) did not correlate to any of the other examinations.

Adolescent↗

Identification of genes induced by neuregulin in cultured myotubes.

The formation of the neuromuscular junction (NMJ) involves a series of inductive interactions between motor neurons and muscle fibers. The neural signals proposed to induce the mRNA expression of acetylcholine receptors in muscle include neuregulin (NRG). In the present study, we have employed RNA fingerprinting by arbitrarily primed PCR analysis to identify the differentially expressed transcripts following NRG treatment in cultured myotubes. Nine partial cDNA fragments were isolated; the mRNA expression of eight of these genes was found to be up-regulated by NRG. The spatial and temporal expression profiles of these NRG-regulated genes in rat tissues during development suggest potential functional roles during the formation of NMJ in vivo. Our findings not only allowed the identification of novel genes, but also suggested possible functions for some known genes that are consistent with their potential roles at the NMJ. Furthermore, the identification of G-protein beta1 subunit and G-protein-coupled receptor as NRG-regulated genes has provided the first demonstration that activation of the NRG signaling pathway can induce the expression of components in the G-protein signaling cascade.

Aging↗

Plant thermal hysteresis proteins.

Proteins which produce a thermal hysteresis (i.e. lower the freezing point of water below the melting point) are common antifreezes in cold adapted poikilothermic animals, especially fishes from ice-laden seas and terrestrial arthropods. However, these proteins have not been previously identified in plants. 16 species of plants collected from northern Indiana in autumn and winter had low levels of thermal hysteresis activity, but activity was absent in summer. This suggests that thermal hysteresis proteins may be a fairly common winter adaptation in angiosperms. Winter stem fluid from the bittersweet nightshade, Solanum dulcamara L., also showed the recrystallization inhibition activity characteristic of the animal thermal hysteresis proteins (THPs), suggesting a possible function for the THPs in this freeze tolerant species. Other potential functions are discussed. Antibodies to an insect THP cross reacted on immunoelectroblots with proteins in S. dulcamara stem fluid, indicating common epitopes in the insect and plant THPs.

Acclimatization↗

Novel optical and statistical methods reveal colloid-wall interactions inconsistent with DLVO and Lifshitz theories.

We present an experimental method based on video microscopy to perform nanometer scale position detection of a micrometer bead in the direction along the propagation of the detection light. Using the same bead for calibration and detection significantly improves the in depth resolution in comparison to video microscopy methods from literature. This method is used together with an optical trap to measure interaction potentials between a glass surface and colloids made of polystyrene or silica at different electrolyte concentrations. The results are confirmed by an independent method where the optical trap is used in connection with a quadrant photodiode. Also, we present a maximum likelihood analysis method which considerably improves the spatial resolution of interaction potentials by optimizing the underlying potential function to fit all observed position distributions. The measured interaction potentials agree well with DLVO theory for small electrolyte concentrations; however, for larger electrolyte concentrations the potentials differ qualitatively from both DLVO and Lifshitz theory.

Journal Article↗

An integrative approach to gain insights into the cellular function of human ataxin-2.

Spinocerebellar ataxia type 2 (SCA2) is a hereditary neurodegenerative disorder caused by a trinucleotide expansion in the SCA2 gene, encoding a polyglutamine stretch in the gene product ataxin-2 (ATX2), whose cellular function is unknown. However, ATX2 interacts with A2BP1, a protein containing an RNA-recognition motif, and the existence of an interaction motif for the C-terminal domain of the poly(A)-binding protein (PABC) as well as an Lsm (Like Sm) domain in ATX2 suggest that ATX2 like its yeast homolog Pbp1 might be involved in RNA metabolism. Here, we show that, similar to Pbp1, ATX2 suppresses the petite (pet-) phenotype of Deltamrs2 yeast strains lacking mitochondrial group II introns. This finding points to a close functional relationship between the two homologs. To gain insight into potential functions of ATX2, we also generated a comprehensive protein interaction network for Pbp1 from publicly available databases, which implicates Pbp1 in diverse RNA-processing pathways. The functional relationship of ATX2 and Pbp1 is further corroborated by the experimental confirmation of the predicted interaction of ATX2 with the cytoplasmic poly(A)-binding protein 1 (PABP) using yeast-2-hybrid analysis as well as co-immunoprecipitation experiments. Immunofluorescence studies revealed that ATX2 and PABP co-localize in mammalian cells, remarkably, even under conditions in which PABP accumulates in distinct cytoplasmic foci representing sites of mRNA triage.

Animals↗

Functional roles of the extracellular segments of the sodium channel alpha subunit in voltage-dependent gating and modulation by beta1 subunits.

Voltage-gated sodium channels consist of a pore-forming alpha subunit associated with beta1 subunits and, for brain sodium channels, beta2 subunits. Although much is known about the structure and function of the alpha subunit, there is little information on the functional role of the 16 extracellular loops. To search for potential functional activities of these extracellular segments, chimeras were studied in which an individual extracellular loop of the rat heart (rH1) alpha subunit was substituted for the corresponding segment of the rat brain type IIA (rIIA) alpha subunit. In comparison with rH1, wild-type rIIA alpha subunits are characterized by more positive voltage-dependent activation and inactivation, a more prominent slow gating mode, and a more substantial shift to the fast gating mode upon coexpression of beta1 subunits in Xenopus oocytes. When alpha subunits were expressed alone, chimeras with substitutions from rH1 in five extracellular loops (IIS5-SS1, IISS2-S6, IIIS1-S2, IIISS2-S6, and IVS3-S4) had negatively shifted activation, and chimeras with substitutions in three of these (IISS2-S6, IIIS1-S2, and IVS3-S4) also had negatively shifted steady-state inactivation. rIIA alpha subunit chimeras with substitutions from rH1 in five extracellular loops (IS5-SS1, ISS2-S6, IISS2-S6, IIIS1-S2, and IVS3-S4) favored the fast gating mode. Like wild-type rIIA alpha subunits, all of the chimeric rIIA alpha subunits except chimera IVSS2-S6 were shifted almost entirely to the fast gating mode when coexpressed with beta1 subunits. In contrast, substitution of extracellular loop IVSS2-S6 substantially reduced the effectiveness of beta1 subunits in shifting rIIA alpha subunits to the fast gating mode. Our results show that multiple extracellular loops influence voltage-dependent activation and inactivation and gating mode of sodium channels, whereas segment IVSS2-S6 plays a dominant role in modulation of gating by beta1 subunits. Evidently, several extracellular loops are important determinants of sodium channel gating and modulation.

Amino Acid Sequence↗

Characterization of the INT6 mammary tumor gene product.

INT6 is a unique gene, highly conserved throughout evolution and associated with mammary tumorigenesis in the mouse. Although it is expressed in all adult tissues of the mouse and early in embryonic development, its function is unknown. To study the normal distribution and the potential function of the Int6 gene products, we produced antibodies against synthetic peptides specific for the Int6 protein. Western blot and immunoprecipitation analysis demonstrated a 43-kD major gene product that is localized in the cytosolic fraction of mammary cell homogenates. This latter observation is supported by immunoperoxidase analysis, which shows a strong staining anti-Int6 peptide in the perinuclear region of the HC11 mammary epithelial cell line, suggesting a possible localization in the Golgi apparatus. Further immunocytochemical studies in the mouse embryo show that Int6 expression is prevalent in migrating neural crest cells, in the notochord, and in condensing cartilage between 9.5 and 14.5 days of development. In these embryonic tissues, Int6 staining co-localizes with the staining of ricinus lectin, and giantin, proteins that are specifically associated with the Golgi apparatus. The restricted expression of the protein within the Golgi apparatus and its strong conservation throughout evolution suggest that Int6 may perform an essential cellular function.

Amino Acid Sequence↗

Phase diagram of water from computer simulation.

The phase diagram of water as obtained from computer simulations is presented for the first time for two of the most popular models of water, TIP4P and SPC/E. This Letter shows that the prediction of the phase diagram is an extremely stringent test for any water potential function, and that it may be useful in developing improved potentials. The TIP4P model provides a qualitatively correct description of the phase diagram, unlike the SPC/E model which fails in this purpose. New behavior not yet observed experimentally is predicted by the simulations: the existence of metastable reentrant behavior in the melting curves of the low density ices (I,III,V) such that it could be possible to transform them into amorphous phases by adequate changes in pressure.

Journal Article↗

An estrogen receptor binding site within the human galanin gene.

Regulation of galanin gene expression in the anterior pituitary (AP) is positively influenced by estrogen in rodents and undetermined in humans. The objective of this study was to investigate the mechanism behind estrogen induction of galanin by identifying any putative estrogen receptor (ER) binding sequences within the human galanin promoter that may function as estrogen response elements (ERE). Two regions, gERE1 and gERE2, were identified in the galanin 5'-flanking sequence with similarity to the full 13-base ERE consensus previously defined in the vitellogenin gene (vERE). Both sequences were tested in mobility shift assays for the ability to bind nuclear proteins isolated from rat AP tissue or MtTW-10 pituitary tumors. Only the distal sequence at -527 (gERE1) yielded an ERE-specific DNA/protein complex distinguished by mobility and cross-competition with vERE. The gel mobility pattern of the DNA/protein complex was comparable between the pituitary tissue and tumor extracts. However, DNA/protein affinity estimations demonstrated a greater affinity of pituitary proteins for gERE1 over the vERE sequence. Evidence that the human ER (hER) does recognize the gERE1 sequence in the human galanin gene was provided by electrophoretic mobility shift assays (EMSAs) with Sf9 extracts enriched in recombinant hER. In addition, antibodies specific for the hER recognized the gERE1/protein complex in supershift experiments. Enhancer activity by gERE1 was detected in transient transfections of the rat GH3 pituitary cell line, resulting in a 4-fold induction of expression driven by the heterologous thymidine kinase promoter in the presence of estrogen. Evidence for ER regulation of the gERE1 enhancer was demonstrated by: 1) inhibition of enhancement using the specific ER antagonist ICI 164,384; and 2) enhancement in HeLa cells that was dependent upon coexpression with hER. Enhancement by gERE1 was half the magnitude as that from the vERE element and may reflect a difference in affinity or composition of the ER complex between the two sequences. These data demonstrate the presence of a functional ERE sequence within the human galanin gene that could potentially function as a regulatory element for estrogen action in the AP.

Animals↗

Heterochromatin and ND10 are cell-cycle regulated and phosphorylation-dependent alternate nuclear sites of the transcription repressor Daxx and SWI/SNF protein ATRX.

Placing regulatory proteins into different multiprotein complexes should modify key cellular processes. Here, we show that the transcription repressor Daxx and the SWI/SNF protein ATRX are both associated with two intranuclear domains: ND10/PML bodies and heterochromatin. The accumulation of ATRX at nuclear domain 10 (ND10) was mediated by its interaction with the N-terminus of Daxx. Binding of this complex to ND10 was facilitated by the interaction of the Daxx C-terminus with SUMOylated promyelocytic leukemia protein (PML). Although ATRX was present at heterochromatin during the entire cell cycle, Daxx was actively recruited to this domain at the end of S-phase. The FACT-complex member structure-specific recognition protein 1 (SSRP1) accumulated at heterochromatin simultaneously with Daxx and accumulation of both proteins depended on ATRX phosphorylation. Both Daxx and SSRP1 were released from heterochromatin early in G(2) phase and Daxx was recruited back to ND10, indicating that both proteins localize to heterochromatin during a very short temporal window of the cell cycle. ATRX seems to assemble a repression multiprotein complex including Daxx and SSRP1 at heterochromatin during a specific stage of the cell cycle, whereas Daxx functions as an adapter for ATRX accumulation at ND10. A potential functional consequence of Daxx accumulation at heterochromatin was found in the S- to G(2)-phase transition. In Daxx(-/-) cells, S-phase was accelerated and the propensity to form double nuclei was increased, functional changes that could be rescued by Daxx reconstitution and that might be the basis for the developmental problems observed in Daxx knockout animals.

Animals↗

Chondrogenic differentiation of mesenchymal stem cells and its clinical applications.

Tissue engineering has the potential to provide cartilaginous constructs capable of restoring the normal function of native articular cartilage following joint injury or degradation. One approach to functional tissue engineering of cartilage involves the in vitro cultivation of tissue constructs by using: (i) chondrogenic cells that can be selected, expanded, and transfected to overexpress the genes of interest, (ii) scaffolds that provide a defined three-dimensional structure for tissue development and biodegrade at a controlled rate. Understanding the functional potential of the cells and the signaling mechanisms underlying their differentiation should lead to innovative protocols for clinical orthopaedic interventions. A large number of growth factors and hormones have been implicated in the regulation of chondrocyte biology, relatively little is known about the intracellular signaling pathways involved. We have tried to define the roles of specific TGF- dependent signaling pathways involved in the regulation of chondrogenesis from human mesenchymal stem cells. Chondrogenesis induced by TGF-beta3 in alginate bead system was confirmed by examining cartilage specific type II collagen expression and aggrecan, whereas type I collagen expression was not affected by TGF-beta3. Type II collagen mRNA expression was expressed strongly during chondrogenesis and MEK inhibition (U0126) resulted in complete down-regulation of type II collagen. In contrast, aggrecan expression was detected in same level by treatment of U0126. These results strongly suggest that the ERK signaling cascade is involved in TGF-beta3 induced-chondrogenesis signaling pathways and a role of its pathway is necessary over a longer period to promote type II collagen expression. However, their end product properties in vivo have not been well known. In this study, an articular cartilage from chondrogenic MSCs with PLGA scaffolds (75:25 and 65:35) were made and analyzed its biochemical, histological and mechanical properties in vitro and in vivo. And also, we evaluated the cartilage formation in vivo through the injection of cell-thermosensitive gel complex, a newly developed injectable material. At 12 weeks after PLGA scaffolds containing chondrogenic MSCs transplantation, the separated rabbit distal femur showed a good gross articular cartilage appearance in the transplanted site. In indentation test, compare to the native articular cartilage, the engineered cartilage from two types of (75:25 and 65:35) achieved up to 30-60% in mechanical stiffness. And also, a new model for cartilage formation in bladder, at 14 weeks after injection, we could find out mass formation in the submucosal area grossly. H&E staining, alcian blue staining and other special staining confirmed the chondrogenic differentiation in the mass. These cell therapy technologies can provide the possibility of clinical applications for vesicoureteral reflux and reflux esophagitis, and urinary incontinence as well as articular cartilage regeneration.

Animals↗