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PubMed1988

Apparent ornithine decarboxylase activity, measured by 14CO2 trapping, after frozen storage of rat tissue and rat tissue supernatants.

Ornithine decarboxylase (ODC) activity of rat tissues was measured by the standard 14CO2 trapping method after frozen storage (-60 or -70 degrees C) of the tissues or their 105,000g supernatants. True ODC activity was determined by two methods: (a) addition of the inhibitors alpha-difluoromethylornithine (DFMO), a specific irreversible inhibitor of ODC, or aminooxyacetate (AOA), an inhibitor that blocks the decarboxylation of ornithine by mitochondrial enzymes; and (b) chromatographic analysis of the reaction products. In the frozen supernatants of liver and spleen, ODC activity changed only slightly after 1 day but increased 29 and 14%, respectively, by 30 days; activity in kidney supernatant decreased 17% after 1 day and remained near that level at 30 days. Kidney and spleen ODC activity was inhibited 90-100% by DFMO, but apparent liver ODC activity was inhibited only 60-75%. In the supernatant prepared from tissue stored frozen for 1 day, apparent ODC activity in liver increased 500% over that activity in the freshly prepared supernatant; at 23 days, apparent activity increased 755% for liver and 121% for kidney. After 23 days, DFMO did not inhibit apparent ODC activity in supernatants from frozen liver and inhibited ODC in frozen kidney by only 49%. With AOA, the ODC activities of the fresh and frozen supernatants were similar, indicating that the large increase in apparent ODC activity in frozen tissue was due to artifacts from the metabolism of ornithine via the mitochondrial pathway. HPLC analysis of the reaction products resulting from the incubation of uniformly labeled [14C]ornithine with the fresh and frozen preparations indicated no increase in putrescine with the frozen preparation.(ABSTRACT TRUNCATED AT 250 WORDS)

Aminooxyacetic Acid↗
PubMed2002

Activation of the alternative complement pathway by fungal melanins.

Melanins are complex biological pigments formed by the oxidative polymerization of phenolic and/or indolic compounds. These pigments have been implicated in the pathogenesis of some microbial infections, malignancies, degenerative disorders, and autoimmune diseases. Recent studies have demonstrated that melanins have antigenic and anti-inflammatory properties. These findings led us to further explore the interaction of melanins with the immune system. Melanin particles ("ghosts") were isolated from in vitro-melanized Cryptococcus neoformans cells and Aspergillus niger conidia and then incubated in normal human serum containing (125)I-labeled complement C3. The results demonstrated deposition of C3 fragments onto the melanin ghosts as early as 1 min after incubation, with maximum deposition occurring after 12 min for C. neoformans-derived melanin ghosts and after 25 min for A. niger-derived melanin ghosts. The blocking of classical pathway activation did not affect the kinetics or total deposition of C3 onto the melanin ghosts, indicating that melanins activate complement through the alternative pathway. Immunofluorescence analysis of lungs from BALB/c mice injected intratracheally with C. neoformans-derived melanin ghosts demonstrated deposition of C3 fragments onto the ghosts. Small granulomas were also observed surrounding the ghosts. However, melanization of the C. neoformans cell wall did not alter the kinetics or total deposition of C3 fragments onto the fungal cells. The finding that melanin surfaces can activate the complement system suggests a potential mechanism for the pathogenesis of some degenerative and/or autoimmune processes that involve melanized cells as well as another potential role for melanin in the virulence of melanin-producing microorganisms.

Animals↗
PubMed2026

Genetic Heterogeneity of Inborn Errors of Immunity Revealed by Whole-Genome Sequencing: Insights from a Russian Patient Cohort.

Identifying genetic cause(s) is a key step for management and treatment of patients with inborn errors of immunity (IEI). Here, in an observational cross-sectional genomic study, we analyzed whole-genome sequencing (WGS) data of 72 IEI patients from Saint Petersburg and Northwestern Russia: 42 patients with common variable immunodeficiency (CVID)-like phenotypes, 6 patients with clinically diagnosed X-linked agammaglobulinemia (XLA or Bruton's disease), and 24 patients with other forms of IEI. Causative pathogenic and likely pathogenic variants in BTK, CYBB, CHD7, AIRE, ATM, SBDS, NFKB1, and CTLA4 genes were identified in 14 (19%) patients. Variants of uncertain significance that could be linked to observed clinical phenotypes were detected in 6 patients. These included a BTK variant in a patient with Bruton's disease, variants in SH2D1A, SOCS1, and IKBKB in patients with CVID, and variants in CARD11 and CD40LG in patients with other forms of IEI. Additional rare variants that were mostly unique to individual patients were found in multiple IEI genes from the International Union of Immunological Societies (IUIS) Expert Committee 2024 list. In the CVID-like subcohort, pathway-level analysis of these rare variants revealed patterns associated with clinical manifestations. Taken together, our results expand the genetic characterization of an understudied regional IEI cohort, particularly of patients with CVID-like phenotypes, and identify genetic factors that are implicated in or may contribute to the disease.

Humans↗
PubMed2025

Proteomics uncovers ICAM2 (CD102) as a novel serum biomarker of proliferative lupus nephritis.

OBJECTIVES: This study aimed to identify novel, non-invasive biomarkers for lupus nephritis (LN) through serum proteomics. METHODS: Serum proteins were detected in patients with LN and healthy control (HC) groups through liquid chromatography-tandem mass spectrometry. The key networks associated with LN were screened out using Cytoscape software, followed by pathway enrichment analysis. The best candidate biomarkers were selected by machine learning models, further validated in a larger independent cohort. Finally, the expression of these candidate markers was verified in kidney tissue samples, and the mechanism was explored by knocking down the expression of intercellular adhesion molecule 2 (ICAM2) through in vitro cell transfection with siRNA. RESULTS: Following the serum proteomic screening of LN, a key network of 20 proteins was identified. Machine learning models were used to select ICAM2 (CD102), metalloproteinase inhibitor 1 (TIMP1) and thrombospondin 1 (THSB1) for validation in independent cohorts. ICAM2 exhibited the highest area under the curve (AUC) value in distinguishing LN from HC (AUC=0.92) and was significantly correlated with activity index, proteinuria, albumin and anti-dsDNA antibody levels. Particularly, ICAM2 was significantly elevated in proliferative LN and was associated with specific pathological attributes, outperforming conventional parameters in distinguishing proliferative LN from non-proliferative LN. ICAM2 expression was also elevated in renal tissue samples from patients with proliferative LN. In vitro, knockdown of ICAM2 expression can inhibit the activation of the PI3K/Akt pathway and alleviate the injury of glomerular endothelial cells. CONCLUSION: ICAM2 (CD102) may serve as a potential serum biomarker for proliferative LN that reflects renal pathology activity, potentially contributing to the progression of LN through the PI3K/Akt pathway.

Humans↗
PubMed2006

CSMD1 is a novel multiple domain complement-regulatory protein highly expressed in the central nervous system and epithelial tissues.

In this study, we describe the identification and in vitro functional activity of a novel multiple domain complement regulatory protein discovered based on its homology to short consensus repeat (SCR)-containing proteins of the regulators of complement activation (RCA) gene family. The rat cDNA encodes a predicted 388-kDa protein consisting of 14 N-terminal CUB domains that are separated from each other by a SCR followed by 15 tandem SCR domains, a transmembrane domain, and a short cytoplasmic tail. This protein is the homolog of the human protein of unknown function called the CUB and sushi multiple domains 1 (CSMD1) protein. A cloning strategy that incorporates the two C-terminal CUB-SCR domains and 12 of the tandem SCR repeats was used to produce a soluble rat CSMD1 protein. This protein blocked classical complement pathway activation in a comparable fashion with rat Crry but did not block alternative pathway activation. Analysis of CSMD1 mRNA expression by in situ hybridization and immunolabeling of neurons indicates that the primary sites of synthesis are the developing CNS and epithelial tissues. Of particular significance is the enrichment of CSMD1 in the nerve growth cone, the amoeboid-leading edge of the growing neuron. These results suggest that CSMD1 may be an important regulator of complement activation and inflammation in the developing CNS, and that it may also play a role in the context of growth cone function.

Aging↗
PubMed1997

Enhancement of spontaneous baroreflex by antisense c-fos oligonucleotide treatment in the NTS of the rat.

We evaluated the hypothesis that basal Fos protein at the nucleus tractus solitarii (NTS), the primary terminal site for baroreceptor afferents, exerts a tonic inhibitory modulation on the spontaneous baroreceptor reflex (BRR) control machinery, which is responsible for beat-to-beat regulation of resting systemic arterial pressure (SAP). In adult male Sprague-Dawley rats anesthetized and maintained with pentobarbital sodium, microinjection bilaterally into the caudal NTS of a 15-mer antisense oligonucleotide that targets against the initiation codon of c-fos mRNA (5'-129 to 143-3') significantly enhanced the spontaneous BRR response, as determined by transfer function analysis of SAP and heart rate signals. The same treatment also diminished baseline Fos-like immunoreactivity in the absence of acute cardiovascular perturbation. Control treatments with artificial cerebrospinal fluid, sense cDNA, or antisense oligonucleotides that either target against a different site of the c-fos mRNA (5'-135 to 149-3') or with three mismatched nucleotides in the antisense sequence, were ineffective. These observations support the notion that, under minimal cardiovascular perturbation, basal expression of Fos protein in the NTS may represent an early step in the cascade of intracellular events that leads to long-term inhibitory modulation of beat-to-beat baroreflex control of blood pressure.

Afferent Pathways↗
PubMed2026

[Laryngeal squamous cell carcinoma-derived exosomes promote neuronal axonal growth by remodeling the neural microenvironment].

Objective: Perineural invasion (PNI) is a critical determinant of poor prognosis in laryngeal squamous cell carcinoma (LSCC), but its underlying mechanisms remain unclear. This study aimed to investigate whether LSCC-derived exosomes induce axonal growth by delivering neuroactive molecules, thereby contributing to tumor perineural invasion. Methods: Clinical data from the laryngeal cancer cohort of The Cancer Genome Atlas Head and Neck Squamous Cell Carcinoma (TCGA-HNSC) dataset were analyzed. Propensity score matching (PSM) and Cox regression were used to evaluate the prognostic value of nerve density, and these findings were validated using 35 pairs of laryngeal cancer and adjacent normal tissue specimens collected at Yantai Yuhuangding Hospital between 2022 and 2026 to assess neural morphological changes. Exosomes were isolated from the human LSCC cell line AMC-HN-8, characterized by quality-control assays, and co-cultured with PC12 cells. A rescue experiment using GW4869, a specific inhibitor of neutral sphingomyelinase, was performed to confirm the exosome-dependent effect. Neurite outgrowth was evaluated by immunofluorescence, and the expression of axonal growth-related genes was measured by RT-qPCR. Targeted metabolomics was employed for the absolute quantification of neuroactive metabolites within the vesicles and for pathway enrichment analysis. Results: After PSM adjustment, high nerve density was identified as an independent poor prognostic factor in LSCC patients (HR=2.10, P=0.035), with particularly pronounced prognostic value in the early-stage node-negative (N0) subgroup (HR=4.07, P=0.001). Pathological sections showed high expression of the neural markers &#x3b2;III-tubulin and PGP9.5 in LSCC tissues (&#x3b2;III-tubulin: t=2.234, P<0.05; PGP9.5: t=2.575, P<0.05). Exosomes were successfully isolated from AMC-HN-8 cells and passed quality control. In vitro assays showed that LSCC-derived exosomes significantly promoted neurite extension and branching in PC12 cells (t=4.147, P<0.000 1) and upregulated core axonal growth genes, including GAP-43, NEFL, and NEFM (GAP-43: t=3.698, P<0.05; NEFL: t=5.113, P<0.01; NEFM: t=5.263, P<0.01); this effect was completely reversed by the exosome-release inhibitor GW4869 (t=3.535, P<0.001). Targeted metabolomics revealed a specific enrichment of 12 neurotransmitters and metabolites within LSCC exosomes, centered on glutamine (83.411 &#x3bc;mol/L, FC=1.88) and glutamate (18.461 &#x3bc;mol/L, FC=1.21), which were significantly enriched in signaling pathways such as "central carbon metabolism in cancer" and "glutamatergic synapse". Conclusion: Nerve density is a potential adverse prognostic factor in patients with LSCC. LSCC-derived exosomes can directly induce axonal growth in neuron-like cells, suggesting that tumor cells actively remodel the neural microenvironment and drive axonal growth through exosome-mediated long-range signaling.

Exosomes↗
PubMed2001

Conditioning of heteronymous H reflex in human temporalis muscle by stimulation of perioral afferents.

A heteronymous H reflex in the temporalis muscle can be elicited by selective stimulation of the masseteric nerve. The present study aimed at defining the optimal amplitude of the H reflex to detect inhibitory changes induced by stimulation of the perioral afferents and at providing new information on the control of masticatory muscles. Sixteen healthy volunteers participated in the experiment. A conditioning stimulus (CS) to the perioral skin was applied at various delays before an ipsilateral selective masseteric nerve stimulation (test stimulus: TS) while the subject was clenching the teeth at 25% of the maximal voluntary contraction. Two intensities of CS and TS were employed, high and low. The peak-to-peak amplitude of the H reflex (TS) and the root-mean-square value of the preceding electromyography were measured and the data analyzed by three-way analysis of variance and Tukey's posthoc tests. For both intensities used the heteronymous H reflex in the temporalis muscle was significantly decreased by prior activation of perioral afferents for delays from 5 to 60 ms. With a delay of 5 and 35 ms the preceding EMG level was not changed, while it was reduced at 20 and 60 ms delay. The intensities used to elicit the heteronymous H reflex of the temporalis muscle were appropriate to detect a reduction in motoneuron excitability. The reduction in the H reflex without a change in the preceding EMG at 5 and 35 ms delays could be due to presynaptic inhibition of the masseteric afferents exerted by the ipsilateral perioral afferents.

Adult↗
PubMed2004

Multiple-probe analysis of folding and unfolding pathways of human serum albumin. Evidence for a framework mechanism of folding.

The changes in the far-UV CD signal, intrinsic tryptophan fluorescence and bilirubin absorbance showed that the guanidine hydrochloride (GdnHCl)-induced unfolding of a multidomain protein, human serum albumin (HSA), followed a two-state process. However, using environment sensitive Nile red fluorescence, the unfolding and folding pathways of HSA were found to follow a three-state process and an intermediate was detected in the range 0.25-1.5 m GdnHCl. The intermediate state displayed 45% higher fluorescence intensity than that of the native state. The increase in the Nile red fluorescence was found to be due to an increase in the quantum yield of the HSA-bound Nile red. Low concentrations of GdnHCl neither altered the binding affinity of Nile red to HSA nor induced the aggregation of HSA. In addition, the secondary structure of HSA was not perturbed during the first unfolding transition (<1.5 m GdnHCl); however, the secondary structure was completely lost during the second transition. The data together showed that the half maximal loss of the tertiary structure occurred at a lower GdnHCl concentration than the loss of the secondary structure. Further kinetic studies of the refolding process of HSA using multiple spectroscopic techniques showed that the folding occurred in two phases, a burst phase followed by a slow phase. An intermediate with native-like secondary structure but only a partial tertiary structure was found to form in the burst phase of refolding. Then, the intermediate slowly folded into the native state. An analysis of the refolding data suggested that the folding of HSA could be best explained by the framework model.

Bilirubin↗
PubMed1993

Voltage dependence of the rat chorda tympani response to Na+ salts: implications for the functional organization of taste receptor cells.

1. Voltage-clamp and current-clamp data were obtained from a circumscribed region of the anterior rat lingual epithelium while simultaneously monitoring the afferent, stimulus-evoked, neural response from the same receptive field. 2. Chorda tympani (CT) responses at constant Na(+)-salt concentration were enhanced by submucosa negative voltage clamp and suppressed by positive voltage clamp. The complete CT response profile, including the time course of adaptation, was not uniquely determined by NaCl concentration alone. The response could be reproduced at different NaCl concentrations by applying a compensating voltage. 3. The form of the concentration and voltage dependence of the CT response indicates that the complete stimulus energy is the Na+ electrochemical potential difference across receptor cell apical membranes, and not Na+ concentration alone. This is the underlying principal behind the equivalence of chemical and electric taste for Na+ salts. 4. CT responses to sodium gluconate (25 and 200 mM) and 25 mM NaCl produced amiloride-insensitive components (AIC) of low magnitude. NaCl at 200 mM produced a significantly larger AIC. The AIC was voltage-clamp independent. The relative magnitude of the AIC was positively correlated with the transepithelial conductance of each salt. This suggests that the large AIC for 200 mM NaCl results from its relatively high permeability through the paracellular pathway. 5. Analysis of the CT response under voltage clamp revealed two anion effects on Na(+)-salt taste, both of which act through the paracellular shunt. 1) Anions modify the transepithelial potential (TP) across tight junctions and thereby modulate the cell receptor potential. This anion effect can be eliminated by voltage clamping the TP. 2) Sufficiently mobile anions facilitate electroneutral diffusion of Na+ salts through tight junctions. This effect is observed especially when Cl- is the anion and when the stimulus concentration favors NaCl influx, allowing Na+ to stimulate receptor cells from the submucosal side. Because the submucosal intercellular spaces are nearly isopotential regions, this effect is insensitive to voltage clamp of the TP. The large AIC associated with this anion effect is due to the low permeability of amiloride.

Afferent Pathways↗
PubMed2000

Retinoid signaling is required to complete the vertebrate cardiac left/right asymmetry pathway.

Vitamin A-deficient (VAD) quail embryos have severe abnormalities, including a high incidence of reversed cardiac situs. Using this model we examined in vivo the physiological function of vitamin A in the left/right (L/R) cardiac asymmetry pathway. Molecular analysis reveals the expression of early asymmetry genes activin receptor IIa, sonic hedgehog, Caronte, Lefty-1, and Fgf8 to be unaffected by the lack of retinoids, while expression of the downstream genes nodal-related, snail-related (cSnR), and Pitx2 is altered. In VAD embryos nodal expression in left lateral plate mesoderm (LPM) is severely downregulated and the expression domain altered during neurulation. Similarly, the expression of cSnR in the right LPM and of Pitx2 in the left side posterior heart-forming region (HFR) is downregulated in the VAD embryos. The lack of retinoids does not cause randomization or ectopic expression of nodal, cSnR, or Pitx2. At the six- to eight-somite stage nodal is expressed transiently in the left posterior HFR of normal quail embryos; this expression is missing in VAD embryos and may be linked to the loss of Pitx2 expression in this region of VAD quail embryos. Administration of retinoids to VAD embryos prior to the six-somite stage rescues the expression of nodal, cSnR, and Pitx2 as well as the randomized VAD cardiac phenotype. There is an absolute requirement for retinoids at the four- to five-somite developmental window for cardiogenesis and cardiac L/R specification to proceed normally. We conclude that retinoids do not regulate the left/right-specific sidedness assignments for expression of genes on the vertebrate cardiac asymmetry pathway, but are required during neurulation for the maintenance of adequate levels of their expression and for the development of the posterior heart tube and a loopable heart. Cardiac asymmetry may be but one of several critical events regulated by retinoid signaling in the retinoid-sensitive developmental window.

Activin Receptors, Type II↗
PubMed2004

Enrichment of presynaptic and postsynaptic markers by size-based gating analysis of synaptosome preparations from rat and human cortex.

BACKGROUND: Synapse regions in the brain are difficult to isolate and study; resealed nerve terminals (synaptosomes) are a widely used in vitro system for the study of neurotransmission, but nonsynaptosomal elements in the homogenate complicate data interpretation. With the goal of quantitative analysis of pathways leading to synapse loss in neurodegenerative disease, we have developed a method that allows focus on the intact synaptosomes within a crude synaptosomal preparation by gating the largest particles based on forward angle light scatter (FSC). METHODS: Crude synaptosomal fractions (P-2) were prepared and labeled with a viability dye (calcein AM), a presynaptic marker (SNAP-25), and a postsynaptic marker (PSD-95). Forward scatter gates based on size standards were drawn to identify the large population (1.4-4.5 microm), and the enrichment of each marker was quantified in preparations from fresh rat homogenates and from cryopreserved human cortex. RESULTS: Gating on forward scatter resulted in an increase that was highly significant (P < 0.001) for all three markers examined. The calcein-AM-positive fraction in the large synaptosomes was 98% +/- 0.8, and 75% +/- 9.8 for rat and human, respectively. Of large particles, 90% +/- 2.7 in rat and 82% +/- 2.6 in human were positive for SNAP-25, indicating a relatively pure population of intact synaptosomes. A total of 76% +/- 2.9 of the large particles were positive for PSD-95 in rat. This compared to 36% +/- 3.0 in human tissue, and indicates that both presynaptic and postsynaptic elements may be analyzed with this methodology. CONCLUSIONS: Most nonsynaptosomal elements can be excluded and the intact subpopulation of interest within the P-2 can be identified based on size. Size-based gating analysis provides a simple and cost-effective method to monitor fluorescence changes in synapse regions.

Aged↗
PubMed1977

Acquisition of the covalent quaternary structure of an immunoglobulin G molecule. Reoxidative assembly in vitro.

We recently reported results of an investigation of the reoxidation of a human, monoclonal immunoglobulin G, following selective reduction of its interchain disulfides by dithiothreitol (Sears, D.W., et al. (1975), Proc. Natl. Acad. Sci. U.S.A. 72, 353). In that work, we described the reoxidative behavior of the molecule under nondissociating conditions. In the present paper, results are presented of the reoxidation of heavy (H) and light (L) chains of this protein alone, or mixed in varying proportions after separation, or mixed with the L chains modified prior to recombination and reoxidation. The overall reoxidative asembly patterns in experiments with H and L separated prior to recombination are similar to those observed when the chains remain noncovalently associated throughout. With equimolar mixtures of H and L, the reoxidation rates also are similar to those of unseparated chains. However, when L chains are present in excess, the overall in vitro rates of covalent assembly are generally diminished, probably indicating transient nonproductive interactions. At the highest molar excesses of L (3:1), the assembly pathways may also be modified. In all experiments with excess L chains, covalent L2 dimers form at rates which are comparatively slow relative to the H2L2 assembly rates. Two kinds of reoxidation experiments with modified L chains are described here for the first time. In the first, the free half-cystine of L is irreversibly blocked by reaction with iodoacetamide, and the alkylated L chains are recombined with reduced H chains. This experiment isolates the reactions in which H2 disulfides are formed without the accompanying formation of HL bonds. Although the alkylated L chains do not play a direct role in the reoxidation, their presence is required to inhibit aggregation and precipitation of high-molecular-weight products which otherwise ensue; this suggests a possible biological role for excess L in vivo. In the second kind of experiment, covalent L2 dimers are mixed with reduced H chains. L2 rapidly disappears with the concurrent appearance of HL, H2L, and fully assembled H2L. H2 dimers are also reactive in this process. Special procedures were developed for analyzing the data from these experiments. A complete format is given for the quantitative determination of the concentration of each of the molecular components directly from spectroscopic scans of the gels. The computational methods solve the general analytical problem posed when staining is not proportional to mass and are applicable to a wide variety of systems utilizing gel electrophoresis to study subunit interactions. A theoretical analysis of pathway and kinetic cooperatively in this system is presented in the following paper (Sears, D.W., and Beychok, S. (1977), Biochemistry 16 (following paper in this issue)).

Bence Jones Protein↗
PubMed2006

Lactosamine modulates the rate of migration of GnRH neurons during mouse development.

Gonadotropin-releasing hormone (GnRH) neurons are derived from progenitor cells in the olfactory placodes and migrate from the vomeronasal organ (VNO) across the cribriform plate into the forebrain. At embryonic day (E)12 in the mouse most of these neurons are still in the nasal compartment but by E15 most GnRH neurons have migrated into the forebrain. Glycoconjugates with carbohydrate chains containing terminal lactosamine are expressed by neurons in the main olfactory epithelium and in the VNO. One of the key enzymes required to regulate the synthesis and expression of lactosamine, beta1,3-N-acetylglucosaminyltransferase-1 (beta3GnT1), is strongly expressed by neurons in the olfactory epithelium and VNO, and on neurons migrating out of the VNO along the GnRH migratory pathway. Immunocytochemical analysis of lactosamine and GnRH in embryonic mice reveals that the percentage of lactosamine+-GnRH+ double-labeled neurons decreases from > 80% at E13, when migration is near its peak, to approximately 30% at E18.5, when most neurons have stopped migrating. In beta3GnT1-/- mice, there is a partial loss of lactosamine expression on GnRH neurons. Additionally, a greater number of GnRH neurons were retained in the nasal compartment of null mice at E15 while fewer GnRH neurons were detected later in embryonic development in the ventral forebrain. These results suggest that the loss of lactosamine on a subset of GnRH neurons impeded the rate of migration from the nose to the brain.

Amino Sugars↗
PubMed2001

Rod pathways in the mammalian retina use connexin 36.

Many neurons in the mammalian retina are coupled by means of gap junctions. Here, we show that, in rabbit retina, an antibody to connexin 36 heavily labels processes of AII amacrine cells, a critical interneuron in the rod pathway. Image analysis indicates that Cx36 is primarily located at dendritic crossings between overlapping AII amacrine cells. This finding suggests that Cx36 participates in homotypic gap junctions between pairs of AII amacrine cells. Cx36 was also found at AII/cone bipolar contacts, previously shown to be gap junction sites. This finding suggests that Cx36 participates at gap junctions that may be heterotypic. These results place an identified neuronal connexin in the context of a well-defined retinal circuit. The absence of Cx36 in many other neurons known to be coupled suggests the presence of additional unidentified connexins in mammalian neurons. Conversely, Cx36 labeling in other regions of the retina is not associated with AII amacrine cells, indicating some other cell types use Cx36.

Animals↗
PubMed2003

Analysis of the eye developmental pathway in Drosophila using DNA microarrays.

Pax-6 genes encode evolutionarily conserved transcription factors capable of activating the gene-expression program required to build an eye. When ectopically expressed in Drosophila imaginal discs, Pax-6 genes induce the eye formation on the corresponding appendages of the adult fly. We used two different Drosophila full-genome DNA microarrays to compare gene expression in wild-type leg discs versus leg discs where eyeless, one of the two Drosophila Pax-6 genes, was ectopically expressed. We validated these data by analyzing the endogenous expression of selected genes in eye discs and identified 371 genes that are expressed in the eye imaginal discs and up-regulated when an eye morphogenetic field is ectopically induced in the leg discs. These genes mainly encode transcription factors involved in photoreceptor specification, signal transducers, cell adhesion molecules, and proteins involved in cell division. As expected, genes already known to act downstream of eyeless during eye development were identified, together with a group of genes that were not yet associated with eye formation.

Animals↗
PubMed2005

Targeted discovery tools: proteomics and chromatin immunoprecipitation-on-chip.

Despite the availability of several completely sequenced genomes, we are still, for the most part, ignorant about how genes interact and regulate each other within a given cell type to specify identity, function and cellular memory. A realistic model of cellular regulation based on current knowledge indicates that many interacting networks operate at the epigenetic, transcriptional, translational and post-translational levels, with feedback between the various levels. Protein-protein and protein-DNA interactions help to define which genes may be activated in a particular cell, and determine whether external cues cause activation or repression. New technologies, e.g. proteomics using mass spectrometry, high-density DNA or oligonucleotide microarrays (chips), and chromatin immunoprecipitation (ChIP), provide new and exciting tools for deciphering the pathways and proteins controlling gene expression. Analysis of these pathways offers new insight that aids targeted drug development.

Chromatin Immunoprecipitation↗
PubMed1993

The expression of deafferentation dysesthesias reduced by dorsal root entry zone lesions in the rat.

Extensive longitudinal lesions of the dorsal root entry zone (DREZ) are effective in relieving some chronic deafferentation pain in humans. A deafferentation syndrome follows C5-T2 dorsal root ganglionectomies in rats. The syndrome consists of biting and scratching the completely and partially denervated limb areas, respectively. This study examines the effect of DREZ lesions on the deafferentation syndrome in the rat. Of 37 rats, 24 underwent C5-T2 ganglionectomies only, five received C4-T3 micromechanical DREZ lesions only, and eight underwent ganglionectomies plus simultaneous DREZ lesions. The animals were observed for 45 days post-operatively. Histological analysis of the spinal cord lesions was performed. All rats with ganglionectomies alone exhibited the deafferentation syndrome; however, no rats with DREZ lesions alone showed this feature. Only 25% of rats with combined ganglionectomies and DREZ lesions exhibited the deafferentation syndrome in the first 30 days, whereas 80% of the animals with ganglionectomies only did so. Although 75% of the animals with combined lesions eventually bit the insensitive forepaw, this behavior was significantly attenuated: the day of onset was delayed and the extent of self-mutilation was reduced. Postmortem histological examination of the DREZ lesions indicated a close association between the completeness of the dorsal horn destruction and the reduction or prevention of self-mutilation. These data support the validity of the animal model and also the hypothesis stating that the deafferentation syndrome results from abnormal spontaneous neural activity in the dorsal horn. Moreover, the variability of the histological findings in these experiments stresses the importance of making contiguous and complete dorsal horn lesions in human DREZ surgery.

Afferent Pathways↗