Diaminobenzidine Reaction Research Articles

A robust method for particulate detection of a genetic tag for 3D electron microscopy.

Genetic tags allow rapid localization of tagged proteins in cells and tissues. APEX, an ascorbate peroxidase, has proven to be one of the most versatile and robust genetic tags for ultrastructural localization by electron microscopy (EM). Here, we describe a simple method, APEX-Gold, which converts the diffuse oxidized diaminobenzidine reaction product of APEX into a silver/gold particle akin to that used for immunogold labelling. The method increases the signal-to-noise ratio for EM detection, providing unambiguous detection of the tagged protein, and creates a readily quantifiable particulate signal. We demonstrate the wide applicability of this method for detection of membrane proteins, cytoplasmic proteins, and cytoskeletal proteins. The method can be combined with different EM techniques including fast freezing and freeze substitution, focussed ion beam scanning EM, and electron tomography. Quantitation of expressed APEX-fusion proteins is achievable using membrane vesicles generated by a cell-free expression system. These membrane vesicles possess a defined quantum of signal, which can act as an internal standard for determination of the absolute density of expressed APEX-fusion proteins. Detection of fusion proteins expressed at low levels in cells from CRISPR-edited mice demonstrates the high sensitivity of the APEX-Gold method.

Detecting Horseradish Peroxidase-Labeled Cells.

A range of substrates is available for detection of cells labeled with horseradish peroxidase (HRP). Diaminobenzidine (DAB) is the most commonly used substrate and one of the most sensitive. It yields an intense brown product that is insoluble in both water and alcohol. DAB staining is compatible with a wide range of common histological stains. DAB can be made more sensitive by adding metal salts such as cobalt or nickel to the substrate solution. The reaction product is slate gray to black, and the products are stable in both water and alcohol. DAB/metal staining is compatible with a wide range of common histological stains. Alternatively, chloronaphthol gives a blue-black product and aminoethylcarbazole (AEC) yields a red product. These substrates are less sensitive than DAB and the products are soluble in alcohol, but they can be used if the DAB reaction gives too high a background or if alternative product colors are required.

Scanning transmission electron microscopic analysis of nitrogen generated by 3, 3'-diaminobenzidine-besed peroxidase reaction with resin ultrathin sections of rhinoceros parotid gland acinar cells.

A 3, 3'-diaminobenzidine (DAB)-based method was used to detect the localization of endogenous peroxidase activity in Indian rhinoceros (Rhinoceros unicornis) parotid gland acinar cells. The tissue had previously been resin-embedded in gelatin capsules for routine electron microscopic observations and thus pre-incubation for endogenous peroxidase analysis was not possible. We attempted to demonstrate the relationship between secretory granules (SGs) in resin ultrathin sections of Indian rhinoceros parotid gland acinar cells and endogenous peroxidase activity. A JEM 1400 Plus scanning transmission electron microscope (STEM) was used to conduct energy dispersive X-ray spectroscopy (EDS) analysis of the presence of nitrogen generated by the DAB reaction in bipartite structural SG consisting of a dense body (or core). The mapping patterns of nitrogen were restricted to the dense body. We observed nitrogen localized in the rough endoplasmic reticulum (ER), nuclear envelope (NE) and several components of the Golgi apparatus (G) of rhinoceros parotid gland acinar cells participating in the synthetic pathway of secretory proteins. Moreover, we established a nitrogen-detection method by EDS analysis of rhinoceros parotid gland. The reliability of the method was validated by comparison of the test group (peroxidase detection in ultrathin resin sections) and the control group (ordinary peroxidase detection in semi-thin sections following glutaraldehyde pre-fixation) of rat submandibular gland. The same mapping patterns of nitrogen were detected by DAB reaction in the SG, ER, NE and G in these two groups. Hence, EDS-STEM approaches for endogenous peroxidase post-incubation analysis will prove useful for advanced cytochemical analysis for the identification of any other resin sections.

Cytochemical Localization of Polyphenol Oxidase Activity in K2-Bodies of Saprolegnia ferax Secondary Zoospores.

Zoospores of the oomycete Saprolegnia ferax release adhesive material from K-bodies at the onset of attachment to substrates. To understand more fully how K-bodies function in adhesion, enzyme activity was investigated cytochemically in secondary zoospores. Presence of catalase, a marker enzyme for microbodies, was explored in the diaminobenzidine (DAB) reaction. Although pH 9.2 DAB-staining characteristic of catalase activity was detected in the granular matrix regions of K-bodies, reaction controls indicated that the reaction was due to oxidative enzyme activity other than catalase. Because polyphenol oxidase (PPO) is another metal-containing enzyme capable of oxidizing DAB, activity of this enzyme was tested with a more specific substrate, dihydroxyphenylalanine (DOPA). In the DOPA procedure, reaction product was exclusively localized within K-bodies, indicating the presence of PPO. Results with three methods of reaction controls (elimination of substrate, addition of a PPO enzyme inhibitor, and heat-inactivation of enzymes) all supported the presence of PPO in K-bodies. This study highlights potential roles for K-body PPO in stabilization of adhesion bodies by: cross-linking matrix phenolic proteins or glycoproteins as K-bodies discharge adhesives onto substrates, or polymerizing phenolics protective against microbial attacks of the adhesion pad.

Morphological Characteristics of a Rat Model of Reexpansion Pulmonary Edema

Objective: Reexpansion pulmonary edema (RPE) is a severe disorder, and its pathophysiology is not well understood. One proposed mechanism for RPE is that chemical substances such as cytokines increase alveolar permeability. Another possible mechanism is that alveolar distention during reexpansion causes physical damage. To test the hypothesis that sudden alveolar distention damages alveolar cellular structure and identify the underlying cause of RPE, we developed and evaluated the morphological characteristics of a rat RPE model. Methods: Lung from a rat model of RPE was observed by using live imaging from intravital fluorescence microscopy with fluorescein isothiocyanate labelled albumin as tracer, light microscopy, and electron microscopy (with and without horseradish peroxidase [HRP] as tracer). Results: Intravital fluorescence microscopy and light microscopy showed that RPE developed almost immediately after lung reexpansion and that blood flow in pulmonary capillaries slowed substantially. In some capillaries, however, blood flow had stopped entirely; in others, anterograde and retrograde flow alternated. Electron microscopy revealed pores in type I pneumocytes and overt fissures in alveolar walls. Electron microscopic observation with HRP revealed that HRP moved from capillaries to the inner surfaces of alveolar epithelia in reexpanded lungs. In addition, diaminobenzidine reaction products from the HRP enzyme reaction were visible in areas with pores. Conclusions: RPE occurred almost immediately after lung reexpansion. Pores developed in type I pneumocytes in alveolar epithelium. These pores, together with overt fissures in alveolar walls, allowed leakage of plasma components into alveoli. These findings appear to be important features of RPE development.

Computer‐aided molecular pathology interpretation in exploring prospective markers for oral submucous fibrosis progression

Evaluation of molecular pathology markers using a computer-aided quantitative assessment framework would help to assess the altered states of cellular proliferation, hypoxia, and neoangiogenesis in oral submucous fibrosis and could improve diagnostic interpretation in gauging its malignant potentiality. Immunohistochemical (IHC) expression of c-Myc, hypoxia-inducible factor-1-alpha (HIF-1α), vascular endothelial growth factor (VEGF), VEGFRII, and CD105 were evaluated in 58 biopsies of oral submucous fibrosis using computer-aided quantification. After digital stain separation of original chromogenic IHC images, quantification of the diaminobenzidine (DAB) reaction pattern was performed based on intensity and extent of cytoplasmic, nuclear, and stromal expression. Assessment of molecular expression proposed that c-Myc and HIF-1α may be used as strong screening markers, VEGF for risk-stratification and VEGFRII and CD105 for prognosis of precancer into oral cancer. Our analysis indicated that the proposed method can help in establishing IHC as an effective quantitative immunoassay for molecular pathology and alleviate diagnostic ambiguities in the clinical decision process.

Unique structures of organelles observed in primary spermatocytes after micro-injection of protein solutions such as immunoglobulin into the lumen of the seminiferous tubules in mice and rats

Unique membranous structures of intracytoplasmic organelle, sting of a stack of a few flat cisternae about 50nm in thickness, were found in mouse and rat spermatocytes after micro-injection of immunoglobulin G into the lumina of the seminiferous tubules. Other proteins such as BSA and cytochrome c used in this study also induced the structures. In most cases, the stacks of cisternae were rolled up like cigars or cylinders. The structures varied in length and diameter, the largest one observed in this study being 10.7μm in length. The structures did not appear when the testes were fixed just after micro-injection and were formed transiently: they were observed in the spermatocytes fixed between 1 and 4h after injection. Cytochrome c, micro-injected as an inter-cellular tracer, was visualised by a diaminobenzidine reaction. As the reaction product was not contained in the cisternae of the unique structures, the lumen of the cisternae of the organelles was not continuous with the inter-cellular space. A flocculent material of low density was observed in the cisternae of the organelle. Similar material was observed in the lumina of solitary cisternae of the rough endoplasmic reticulum in the spermatocytes, suggesting that the structures derived from endoplasmic reticulum.

Visualization of ATP with Luciferin-Luciferase Reaction in Mouse Skeletal Muscles Using an “In Vivo Cryotechnique”

Adenosine triphosphate (ATP) is a well-known energy source for muscle contraction. In this study, to visualize localization of ATP, a luciferin-luciferase reaction (LLR) was performed in mouse skeletal muscle with an "in vivo cryotechnique" (IVCT). First, to confirm if ATP molecules could be trapped and detected after glutaraldehyde (GA) treatment, ATP was directly attached to glass slides with GA, and LLR was performed. The LLR was clearly detected as an intentional design of the ATP attachment. The intensity of the light unit by LLR was correlated with the concentration of the GA-treated ATP in vitro. Next, LLR was evaluated in mouse skeletal muscles with IVCT followed by freeze-substitution fixation (FS) in acetone-containing GA. In such tissue sections the histological structure was well maintained, and the intensity of LLR in areas between muscle fibers and connective tissues was different. Moreover, differences in LLR among muscle fibers were also detected. For the IVCT-FS tissue sections, diaminobenzidine (DAB) reactions were clearly detected in type I muscle fibers and erythrocytes in capillaries, which demonstrated flow shape. Thus, it became possible to perform microscopic evaluation of the numbers of ATP molecules in the mouse skeletal muscles with IVCT, which mostly reflect living states.

Altered expression of FosB/ΔFosB in central autonomic regions in hypertensive obese Zucker rats

Adult obese Zucker rats (OZR) have elevated sympathetic nerve activity and blood pressure vs. lean Zucker rats (LZR). We determined whether autonomic regulatory regions in the brain are chronically activated in OZR vs. LZR by immunohistochemical examination of FosB/ΔFosB expression. Male rats (14 wks) were perfused with paraformaldehyde and brains were sectioned coronally. One in six sections were processed for FosB expression (Santa Cruz goat anti‐FosB) via a nickel diaminobenzidine (DAB) reaction. A subset was processed to reveal catecholaminergic neurons (Chemicon mouse anti‐tyrosine hydroxylase) via a DAB reaction. In nucleus tractus solitarius (−13.1 to −13.5 mm bregma) FosB expression was higher (85–134%) in OZR (7) vs. LZR (6) at all levels. In rostral ventrolateral medulla (−12.0 to −12.6 mm bregma) FosB expression was enhanced at the most caudal level (−12.6 mm) in OZR with comparable expression in other levels in OZR (5) vs. LZR (5). In NTS and RVLM there was no FosB expression in catecholaminergic neurons in OZR or LZR. In the hypothalamic paraventricular nucleus (PVN) OZR (5) had less FosB expression (94–200%) vs. LZR (5). Similarly, in the anterior hypothalamic nucleus OZR(6) had less (18–44%) expression than LZR(6). These data suggest that the chronic activations of multiple autonomic regions are altered in OZR vs. LZR, and future studies are needed to identify the phenotypes of the activated neurons.

Low-Dose-Rate, Low-Dose Irradiation Delays Neurodegeneration in a Model of Retinitis Pigmentosa

The existence of radiation hormesis is controversial. Several stimulatory effects of low-dose (LD) radiation have been reported to date; however, the effects on neural tissue or neurodegeneration remain unknown. Here, we show that LD radiation has a neuroprotective effect in mouse models of retinitis pigmentosa, a hereditary, progressive neurodegenerative disease that leads to blindness. Various LD radiation doses were administered to the eyes in a retinal degeneration mouse model, and their pathological and physiological effects were analyzed. LD gamma radiation in a low-dose-rate (LDR) condition rescues photoreceptor cell apoptosis both morphologically and functionally. The greatest effect was observed in a condition using 650 mGy irradiation and a 26 mGy/minute dose rate. Multiple rounds of irradiation strengthened this neuroprotective effect. A characteristic up-regulation (563%) of antioxidative gene peroxiredoxin-2 (Prdx2) in the LDR-LD-irradiated retina was observed compared to the sham-treated control retina. Silencing the Prdx2 using small-interfering RNA administration reduced the LDR-LD rescue effect on the photoreceptors. Our results demonstrate for the first time that LDR-LD irradiation has a biological effect in neural cells of living animals. The results support that radiation exhibits hormesis, and this effect may be applied as a novel therapeutic concept for retinitis pigmentosa and for other progressive neurodegenerative diseases regardless of the mechanism of degeneration involved.

Detection of tissue transglutaminase with new antibodies in immunohistochemistry

Background: Tissue transglutaminase (tTG) is the most abundantly expressed transglutaminase (TG) enzyme in the human brain. There are some indications for the participation of tTG in the development of neurodegenerative diseases, including Alzheimer’s and Parkinson’s disease. In brain, tTG is known to be expressed in neurons and astroglia (Wilhelmus et al. 2008). Previous data showed tTG being involved in cross-linking s-amyloid and tau and therefore contributing to the development of Alzheimer’s disease hallmarks. It has been observed that neurofibrillary tangles and s-amyloid in senile plaques show immunoreactivity for tTG (Wilhelmus et al. 2008). Furthermore it has been described that transglutaminase activity is increased in Alzheimer’s disease (e.g. Wang et al. 2008). Methods: We tested 12 new monoclonal antibodies with specific reactivity to human tTG (Wolf et al. 2011) on tissue samples of AD brains and age-matched controls. tTG immunoreactivity was compared to tau an amyloid pathology. Additionally we compared our results to those of assays for specific determination of either tTG protein or in-vitro activatable tTG made of adequate regions. Results: The monoclonal antibodies against human tTG showed different immunoreactivity in human brain tissue. Some of them show clear distribution patterns, different in AD brains and controls. Concerning AD pathology we observed a high density of tTG in neurofibrillary tangles and a clear colocalization of tTG and anti-phosphotau antibody. In senile plaques only a light shade of tTG immunoreactivity could be detected with nickel enhanced diaminobenzidine reaction. Some of the antibodies detect tTG associated with reactive Astroglia around senile plaques. Conclusions: Some of the new anti-human tTG antibodies clearly visualize distribution and density of substrate-associated tTG in human brain. The further characterization of their specificity and detected epitopes is just in process. In AD brain, the different density of tTG in aggregated tau and s-amyloid may be caused due to the different concentrations of lysine in tau and s-amyloid peptides. Together with the results of quantification of tTG total protein and activity assays, the new antibodies may contribute to further investigation of tTG in human brain and their role in the development of neurodegenerative diseases like AD.

Low‐voltage activated Kv1.1 subunits are crucial for the processing of sound source location in the lateral superior olive in mice

Voltage-gated potassium (Kv) channels containing Kv1.1 subunits are strongly expressed in neurons that fire temporally precise action potentials (APs). In the auditory system, AP timing is used to localize sound sources by integrating interaural differences in time (ITD) and intensity (IID) using sound arriving at both cochleae. In mammals, the first nucleus to encode IIDs is the lateral superior olive (LSO), which integrates excitation from the ipsilateral ventral cochlear nucleus and contralateral inhibition mediated via the medial nucleus of the trapezoid body. Previously we reported that neurons in this pathway show reduced firing rates, longer latencies and increased jitter in Kv1.1 knockout (Kcna1−/−) mice. Here, we investigate whether these differences have direct impact on IID processing by LSO neurons. Single-unit recordings were made from LSO neurons of wild-type (Kcna1+/+) and from Kcna1−/− mice. IID functions were measured to evaluate genotype-specific changes in integrating excitatory and inhibitory inputs. In Kcna1+/+ mice, IID sensitivity ranged from +27 dB (excitatory ear more intense) to −20 dB (inhibitory ear more intense), thus covering the physiologically relevant range of IIDs. However, the distribution of IID functions in Kcna1−/− mice was skewed towards positive IIDs, favouring ipsilateral sound positions. Our computational model revealed that the reduced performance of IID encoding in the LSO of Kcna1−/− mice is mainly caused by a decrease in temporal fidelity along the inhibitory pathway. These results imply a fundamental role for Kv1.1 in temporal integration of excitation and inhibition during sound source localization.

Bright Field Neuronal Preparation Optimized for Automatic Computerized Reconstruction, a Case with Cerebellar Climbing Fibers

To make the best use of computerized automatic reconstruction it is crucial to supply optimized neuronal preparations for reconstruction. For bright field preparations a major technique for labeling a specific population of neurons is the injection of tracers, such as dextran conjugated with biotin (biotinylated dextran amine, BDA). BDA-labeled neurons and axons can then be visualized in brain sections using the diaminobenzidine (DAB) reaction, which causes BDA to form a dark precipitate. It is important for such brain sections to be oriented in an appropriate direction so that labeled neurons and axons are localized within individual sections to the greatest extent possible, because automatic reconstruction can be applied only within single brain sections (thickness of 50–100 μm) at the moment. In this sense, cerebellar climbing fibers, which are organized within a flat longitudinal plane, are among the best suited neuronal elements for automatic reconstruction. For automatic reconstruction, it is also important to separate individual, labeled neurons (or axons) from each other by controlling the amount of tracer. By solving these points the histological preparation can be optimized for automatic computerized reconstruction. Developers and users of automatic reconstruction software are usually different individuals. Mutual understanding of the technical difficulties and scientific needs on either side is indispensable to optimize collaboration between the two groups. We provided bright-field preparation of climbing fibers labeled with BDA to the DIADEM challenge. By looking at how the automatic reconstruction software works in the DIADEM conference, I realized that whether the computerized

Mapping of FcεRI immunoglobulin E receptor in activated mast cells by scanning near-field optical microscopy

Introduction. The cell membrane has a dynamic role that enables the reorganization of receptors and signal molecules in response to signaling processes. To visualize these phenomena we can nowadays benefit from techniques that allow subdiffraction optical resolution.(1) Among these, scanning near-field optical microscopy (SNOM) exploits the evanescent field exiting at the probe fibre apex. The lateral resolution depends essentially on the subwavelength aperture size of the optical fibre (typically better than 100 nm). This makes SNOM particularly suited for nanoscale study on intact biological membrane. Recently, we demonstrated that SNOM combined with immunolabelling and diaminobenzidine (DAB) staining is a valuable non-invasive approach for investigating nanostructures components within intact oligodendrocites.(2) Here we extend this approach to the study of reorganization of FceRI immunoglobulin E (IgE) receptor in intact mast cells upon antigen-induced degranulation by IgE cross-linking. Materials and Methods. Rat basophilic leukemia RBL-2H3 cells were grown on coverslips, incubated overnight with anti-2,4-Dinitrophenol (DNP) IgE and degranulated by adding DNP. After 30’ cells were fixed and immunolabelled with anti-FceRI monoclonal antibody. DAB staining was performed with VECTASTAIN® UNIVERSAL Elite ABC kit. TriA-SNOM microscope (A.P.E. Research, Trieste) was used for near-field measurements. Results and discussions. Resting and activated cells with and without DAB staining immunolabelling for the internal portion of FceRI are analyzed by SNOM. Topography of activated cells shows membrane ridges over surface and in some cases a considerable cell flattening, in general accordance with the morphology observed as result of the degranulation process. The optical transmission images of DAB stained activated mast cell display numerous very dark circular areas, not observed in the unlabelled activated cells. Such features are likely due to a strong light adsorption for the presence of localized DAB reaction. These areas (lateral size about 300 nm) appear to be in agreement with IgE receptor FceRI patches observed on cytoplasmatic side of membrane sheets of activated mast cells.(3) In conclusion these results demonstrate that SNOM combined with immunolabelling and DAB staining holds great potential for investigating the organization of proteins into micro- or nanodomains in cell membrane. 1. Bioch. Biophys. Acta 2010, 1798: 77. 2. Neuroimage 2010, 49: 517. 3. Biophys. J. 2006, 90: 2404.

Pattern of expression of the CREG gene and CREG protein in the mouse embryo

The cellular repressor of E1A-stimulated genes (CREG) is a secreted glycoprotein that inhibits cell proliferation and/or enhances differentiation. CREG is widely expressed in adult tissues such as the brain, heart, lungs, liver, intestines and kidneys in mice. We investigated the level of CREG expression during mouse embryogenesis and its distribution at 18.5 days post coitus (dpc) using immunohistochemical staining with diaminobenzidine, western blotting and reverse transcription-polymerase chain reaction. CREG expression was first detected in mouse embryos at 4.5 dpc. It was expressed at almost all stages up to 18.5 dpc. The level of CREG was found to increase gradually and was highest at 18.5 dpc. Western blotting showed that the CREG protein was expressed at higher levels in the brain, heart, intestines and kidneys than in the lungs and liver at 18.5 dpc. In 9.5 dpc embryos, CREG was expressed only in the endothelial cells of blood vessels, after the vascular lumen had formed. With advanced differentiation, vascular smooth muscle cells developed in the embryonic vascular structures; the expression of smooth muscle α-actin protein and CREG were positive and increased gradually in 10.5 dpc embryonic vessels. CREG expression in the embryonic blood vessels peaked at 15.5 dpc and was reduced slightly at 18.5 dpc. These results indicate that CREG is expressed during mouse embryogenesis and might participate in the differentiation of these organs during embryogenesis.

SENP1 Induces Prostatic Intraepithelial Neoplasia through Multiple Mechanisms

SUMOylation has been shown to modulate DNA replication/repair, cell cycle progression, signal transduction, and the hypoxic response. SUMO (small ubiquitin-like modifier)-specific proteases regulate SUMOylation, but how changes in the expression of these proteases contribute to physiological and/or pathophysiological events remains undefined. Here, we show that SENP1 (sentrin/SUMO-specific protease 1) is highly expressed in human prostate cancer specimens and correlates with hypoxia-inducing factor 1alpha (HIF1alpha) expression. Mechanistic studies in a mouse model indicate that androgen-driven expression of murine SENP1 leads to HIF1alpha stabilization, enhanced vascular endothelial growth factor production, and angiogenesis. Further pathological assessment of the mouse indicates that SENP1 overexpression induces transformation of the normal prostate gland and gradually facilitates the onset of high-grade prostatic intraepithelial neoplasia. Consistent with cell culture studies, SENP1 enhances prostate epithelial cell proliferation via modulating the androgen receptor and cyclin D(1). These results demonstrate that deSUMOylation plays a critical role in prostate pathogenesis through induction of HIF1alpha-dependent angiogenesis and enhanced cell proliferation.

Conjunctival Interleukin-13 Expression in Mucous Membrane Pemphigoid and Functional Effects of Interleukin-13 on Conjunctival Fibroblasts in Vitro

Interleukin-13 (IL-13) is the dominant effector cytokine of fibrosis in pulmonary and liver disease. Excessive conjunctival fibrosis in the immunobullous disease ocular mucous membrane pemphigoid (MMP) causes blindness; the pathogenesis of scarring in this disease is incompletely understood. To determine whether IL-13 is involved in conjunctival fibrosis in MMP, we studied the expression of IL-13 in ocular MMP patients before and after systemic immunosuppression and examined the effects of IL-13 on normal human conjunctival fibroblasts. We found high stromal cell expression of IL-13 in active ocular MMP by immunohistochemistry; 80% of these cells were CD3-positive T cells. Following immunosuppression, in clinically uninflamed, treated, ocular MMP patients, the number of IL-13 positive cells was significantly reduced, but this was still fourfold greater than in normal conjunctiva. IL-13 stimulated collagen lattice contraction and migration, and decreased production of mmp-3 and mmp-10 by human conjunctival fibroblasts. The addition of T cell culture supernatant to IL-13 synergistically augmented fibroblast migration. IL-13 also up-regulated surface expression of HLA-DR, CD80, CD40, and CD154 by conjunctival fibroblasts, suggesting a potential mechanism for fibroblast-T cell cross talk, via which fibroblasts may actively engage in perpetuating chronic inflammation and continued fibrosis. Together, these findings suggest that IL-13 is involved in conjunctival fibrosis in MMP, and that IL-13 has both profibrotic and pro-inflammatory effects on human conjunctival fibroblasts.

The Transcriptional Repressor CcpN from Bacillus subtilis Uses Different Repression Mechanisms at Different Promoters

CcpN, a transcriptional repressor from Bacillus subtilis that is responsible for the carbon catabolite repression of three genes, has been characterized in detail in the past 4 years. However, nothing is known about the actual repression mechanism as yet. Here, we present a detailed study on how CcpN exerts its repression effect at its three known target promoters of the genes sr1, pckA, and gapB. Using gel shift assays under non-repressive and repressive conditions, we showed that CcpN and RNA polymerase can bind simultaneously and that CcpN does not prevent RNA polymerase (RNAP) binding to the promoter. Furthermore, we investigated the effect of CcpN on open complex formation and demonstrate that CcpN also does not act at this step of transcription initiation at the sr1 and pckA and presumably at the gapB promoter. Investigation of abortive transcript synthesis revealed that CcpN acts differently at the three promoters: At the sr1 and pckA promoter, promoter clearance is impeded by CcpN, whereas synthesis of abortive transcripts is repressed at the gapB promoter. Eventually, we demonstrated with Far Western blots and co-elution experiments that CcpN is able to interact with the RNAP alpha-subunit, which completes the picture of the requirements for the repressive action of CcpN. On the basis of the presented results, we propose a new working model for CcpN action.

Caspase-3 Is Enriched in Postsynaptic Densities and Increased in Alzheimer's Disease

Progressive synaptic degeneration and neuron loss are major structural correlates of cognitive impairment in Alzheimer's disease (AD). The mechanisms by which synaptic degeneration in AD occurs have not been established. The activation of proteins within the caspase family has been implicated in AD-associated neurodegeneration, and synaptically localized caspase activity could play a role in the synaptic degeneration and loss found in AD. We used synaptosomal fractionation with Western blotting and immunohistochemistry to examine the anatomical, subcellular, and subsynaptic expression patterns of caspase 3 in both the anterior cingulate cortex and hippocampus of control and AD patients. In both control and AD cases, there was a selective enrichment of caspase- 3 at synapses, particularly in the postsynaptic density (PSD) fractions. Compared with controls, AD patients exhibited significant increases in synaptic procaspase- 3 and active caspase-3 expression levels that were most evident in the PSD fractions. These data demonstrate for the first time the preferential localization and increase of caspase-3 in the PSD fractions in AD and suggest an important role for caspase 3 in synapse degeneration during disease progression.

CGI-58 Is an α/β-Hydrolase within Lipid Transporting Lamellar Granules of Differentiated Keratinocytes

CGI-58 is the causative molecule underlying Dorfman-Chanarin syndrome, a neutral lipid storage disease exhibiting apparent clinical features of ichthyosis. CGI-58, associated with triacylglycerol hydrolysis, has an alpha/beta-hydrolase fold and is also known as the alpha/beta-hydrolase domain-containing protein 5. The purpose of this study was to elucidate the function of CGI-58 and the pathogenic mechanisms of ichthyosis in Dorfman-Chanarin syndrome. Using an anti-CGI-58 antibody, we found CGI-58 to be expressed in the upper epidermis, predominantly in the granular layer cells, as well as in neurons and hepatocytes. Immunoelectron microscopy revealed that CGI-58 was also localized to the lamellar granules (LGs), which are lipid transport and secretion granules found in keratinocytes. CGI-58 expression was markedly reduced in the epidermis of patients with harlequin ichthyosis, demonstrating defective LG formation. In cultured keratinocytes, CGI-58 expression was mildly up-regulated under high Ca(2+) conditions and markedly up-regulated in three-dimensional, organotypic cultures. In the developing human epidermis, CGI-58 immunostaining was observed at an estimated gestational age of 49 days, and CGI-58 mRNA expression was up-regulated concomitantly with both epidermal stratification and keratinocyte differentiation. CGI-58 knockdown reduced expression of keratinocyte differentiation/keratinization markers in cultured human keratinocytes. Our results indicate that CGI-58 is expressed and packaged into LGs during keratinization and likely plays crucial role(s) in keratinocyte differentiation and LG lipid metabolism, contributing to skin lipid barrier formation.