Vesicular Glutamate Transporter 1 (VGLUT1)- and VGLUT2-containing Terminals on the Rat Jaw-closing γ-Motoneurons

Currently, compared to jaw-closing (JC) α-motoneurons, the information on the distribution and morphology of glutamatergic synapses on the jaw-closing (JC) γ-motoneurons, which may help elucidate the mechanism of isometric contraction of the JC muscle, is very limited. This study investigated the distribution and ultrastructural features of vesicular glutamate transporter 1 (VGLUT1)- and VGLUT2-immunopositive (+) axon terminals (boutons) on JC γ-motoneurons by retrograde tracing with horseradish peroxidase, electron microscopic immunocytochemistry, and quantitative analysis. About 35% of the boutons on identified JC γ-motoneurons were VGLUT+, and of those, 99% were VGLUT2+. The fraction of VGLUT1+ boutons of all boutons and the percentage of membrane of JC γ-motoneurons covered by these boutons were significantly lower than those for the JC α-motoneurons, revealed in our previous work. The bouton volume, mitochondrial volume, and active zone area of the VGLUT2+ boutons on the JC γ-motoneurons were uniformly small. These findings suggest that the JC γ-motoneurons, in contrast to the JC α-motoneurons, receive generally weak glutamatergic synaptic input almost exclusively from VGLUT2+ premotoneurons that form direct synapse with motoneurons.

Enzyme-linked immunosorbent assay for detecting anti-pertussis toxin antibody in mouse

PURPOSE: Although the DTaP and Tdap vaccines used to prevent pertussis have been used for a long time, there is no standard method for measuring pertussis antigens. Therefore, this preliminary study was conducted to develop an enzyme-linked immunosorbent assay method using an animal model for measuring antibodies against pertussis toxin, the most important pertussis pathogenic antigen, in the sera of vaccinated mice. MATERIALS AND METHODS: Bordetella pertussis Tohama phase I was cultured for 24–30 hours, and then pertussis toxin was purified from the culture medium by chromatography. Purified pertussis toxin was diluted in phosphate-buffered saline-coating buffer, and 100 µL of diluted pertussis toxin was added to each well and reacted at room temperature for 4 hours. Positive serum was diluted to 1/1,250–1/80,000 and negative serum was diluted to 1/50 to determine the coating concentration with the optimal signal/noise ratio. Optimal test conditions were confirmed from the dilution factors of the secondary antibody and streptavidin horseradish peroxidase (SA-HRP). RESULTS: Optimal conditions were as follows: 4 µg/mL for coating antigen; 1/40,000 for secondary antibody; and 1/1,000 for the SA-HRP dilution factor. Comparison of the sera obtained from mice treated with a developing vaccine and commercial vaccine with National Institute for Biological Standard and Control standard serum under the established conditions showed the following results: 1,300.62, 534.94, and 34.85, respectively. CONCLUSION: The method developed in this study is suitable for measuring anti-pertussis toxin antibodies and may be applicable for clinical sample analysis or indirect diagnosis of pertussis.

L1 Recombinant Proteins of HPV Tested for Antibody Forming Using Sera of HPV Quadrivalent Vaccine

Virus-like particles (VLPs) derived from human papillomavirus (HPV) L1 capsid proteins were used for HPV quadrivalent recombinant vaccine. The HPV quadrivalent vaccine is administrated in a 3-dose regimen of initial injection followed by subsequent doses at 2 and 6 months to prevent cervical cancer, vulvar, and vaginal cancers. The type 6, 11, 16, or 18 of HPV infection is associated with precancerous lesions and genital warts in adolescents and young women. The HPV vaccine is composed of viral L1 capsid proteins are produced in eukaryotic expression systems and purified in the form of VLPs. Four different the L1 protein of 3 different subtypes of HPV: HPV11, HPV16, and HPV18 were expressed in Escherichia coli divided into 2 fragments as N- and C-terminal of each protein in order to examine the efficacy of HPV vaccine. Vaccinated sera failed to recognize N-terminal L1 HPV type 16 and type 18 by western blot while they detected N-terminal L1 protein of HPV type 11. Moreover, the recombinant C-terminal L1 proteins of type 16 was non-specifically recognized by the secondary antibody conjugated with horseradish peroxidase. This expression and purification system may provide simple method to obtain robust recombinant L1 protein of HPV subtypes to improve biochemical analysis of antigens with immunized sera.

Immobilization of horseradish peroxidase on Fe3O4 magnetic nanoparticles

Background: Iron magnetic nanoparticles have attracted much attention. They have been used in enzyme immobilization because of their properties such as product is easily separated from the medium by magnetic separation. The present work was designed to immobilize horseradish peroxidase on Fe3O4 magnetic nanopraticles without modification. Results: In the present study, horseradish peroxidase (HRP) was immobilized on non-modified Fe3O4 magnetic nanoparticles. The immobilized HRP was characterized by FT-IR spectroscopy, scanning electron microscopy, and energy dispersive X-ray. In addition, it retained 55% of its initial activity after 10 reuses. The optimal pH shifted from 7.0 for soluble HRP to 7.5 for the immobilized HRP, and the optimal temperature shifted from 40°C to 50°C. The immobilized HRP is more thermostable than soluble HRP. Various substrates were oxidized by the immobilized HRP with higher efficiencies than by soluble HRP. Km values of the soluble and immobilized HRP were 31 and 45 mM for guaiacol and 5.0 and 7.0 mM for H2O2, respectively. The effect of metals on soluble and immobilized HRP was studied. Moreover, the immobilized HRP was more stable against high concentrations of urea, Triton X-100, and isopropanol. Conclusions: Physical immobilization of HRP on iron magnetic nanoparticles improved the stability toward the denaturation induced by pH, heat, metal ions, urea, detergent, and water-miscible organic solvent.

ELISA for Quantitative Determination of Hepatitis B Virus Surface Antigen

Several studies have reported a good correlation between levels of serum hepatitis B virus surface antigen (HBsAg) and covalently closed circular DNA (cccDNA) before and after antiviral therapy. As a result, the quantification of HBsAg levels has attracted much attention in recent years as an important approach to evaluate viral activity. In this study, mAbs against HBsAg were generated and 9 mAbs (H17, H30, H31, H67, H73, H97, H101, H118, and H128) were investigated for optimization of HBsAg quantitation ELISA. Determination of the best combinations of mAbs for sandwich ELISA identified H17 and H31 mAbs as the ideal capture and horseradish peroxidase (HRP) conjugate mAbs, respectively. A standard curve for the current assay system exhibited linearity up to 40 ng/ml of HBsAg while a detection limit of approximately 1 ng/ml of HBsAg was also estimated, which was comparable to that of the other commercial ELISA kits. The ELISA system established in this study is particularly differentiated from other commercial kits in using mAbs for both capture and HRP conjugate, which provides a solution to inconsistency of quality and ethical issues in polyclonal antibodies production using laboratory animals.

Involvement of cross-reactive carbohydrate determinants-specific IgE in pollen allergy testing

BACKGROUND: Specific IgE antibodies against the low-molecular-weight carbohydrate antigen that does not bridge IgE molecules on mast cells are not associated with clinical symptoms. Cross reactivity can be determined in allergen-specific IgE detection assays when the carbohydrate structures between pollen allergens and plant derived food allergens are similar; in such cases, false positive results for grain or legume allergens can be reported for pollen allergic patients who are not sensitized to those allergens. This phenomenon arises owing to the presence of cross-reactive carbohydrate determinants (CCDs). OBJECTIVE: This study aimed to assess the impact of CCD interference on the results for pollen allergen-specific IgE antibodies in the general adult population and to perform CCD inhibition tests evaluating the involvement of CCD on samples positive to pollen allergens. METHODS: Serum samples from 322 subjects were tested for IgE antibodies to pollens and CCD. The research subjects were given questionnaires about pollen allergic symptoms to help assess the presence of allergies. Allergen IgE antibodies for Japanese cedar, Japanese cypress, orchard grass, ragweed, MUXF, bromelain, horseradish peroxidase (HRP), and ascorbate oxidase (ASOD) were analyzed. RESULTS: It was observed that among individuals who tested positive to any of the pollen allergens, the positive ratio of CCD-specific IgE antibody was the highest for HRP (13.5%–50.0%). The results from the inhibition tests revealed that CCD was marginally present. Although IgE antibodies for cedar pollen did not react with CCD, IgE antibodies for Japanese cypress, orchard grass, and ragweed might be detected by the presence of CCD. CONCLUSION: The results of the inhibition tests revealed the obvious presence of CCD suggesting its involvement. Considering these findings, careful evaluation of patient IgE results should be performed for Japanese cypress, orchard grass, and ragweed.

Production of a conjugate between the rK346 antigen from Leishmania infantum and the horseradish peroxidase C for the detection of rK346 antibodies / Producción de un conjugado entre el antígeno rK346 de Leishmania infantum y la peroxidasa C de rábano picante para la detección de anticuerpos rK346

It was designed and characterized a reporter system to be captured by antibodies bound to ELISA plates. The system was designed with the rK346 from Leishmania infantum, a highly antigenic and specific protein. The rK346 was coupled to the horseradish peroxidase C (HRPc) from Armoracia rusticana using glutaraldehyde or sulfo-SMCC. Glutaraldehyde conjugation was performed in two steps. Separation of conjugates was carried out using a Sepharose S-200 in size exclusion chromatography (SEC); fractions were analyzed via HRPc activity and through ELISA plates sensitized with polyclonal anti-rK346 IgG purified from rabbit serum. A heterogeneous population of conjugates rK346-HRPc was obtained with molecular weights ranging between 109.7 ± 16.5 to 67.6 ± 10.1 kDa; with rK346-HRPc stoichiometries of 1:2; 2:1; 3:1; and 2:2. Conjugation using sulfo-SMCC was carried out first by introducing -SH groups onto the HRPc using the SATA reagent and the antigen was modified with sulfo-SMCC during 45 min. Separation and analysis of conjugates was performed similarly as with glutaraldehyde, resulting in a heterogeneous population of conjugates rK346-HRPc with molecular weights between 150.5 ± 22.6 to 80.0 ± 12.0 kDa; with rK346-HRPC stoichiometries of 2:1; 1:2; 2:2; and 1:3, with an increased conjugation efficiency in comparison with glutaraldehyde. This enables sulfo-SMCC to be used as a potential reagent for coupling the antigen to the HRPc, to design an economic, specific and easy method to apply as a reporter system, available to assess individuals at risk and/or at early and late stages of visceral leishmaniasis.

Se diseñó y caracterizó un sistema reportero para ser capturado por anticuerpos enlazados a placas de ELISA. El sistema fue diseñado con una proteína altamente antigénica y específica, la rK346 de Leishmania infantum. La rK346 fue acoplada a la peroxidasa C de rábano picante (HRPc) de Armoracia rusticana usando glutaraldehido o sulfo-SMCC. La conjugación con glutaraldehido fue realizada en dos pasos. La separación de los conjugados fue llevada a cabo a través de una cromatografía de exclusión molecular sefarosa S-200 (CES), las fracciones fueron analizadas midiendo la actividad HRPc y por placas ELISA sensibilizadas con inmunoglobulina G policlonal anti-rK346, purificada desde suero de conejo. Se obtuvo una población heterogénea de conjugados rK346-HRPc en un rango de pesos moleculares entre 109,7 ± 16,5 a 67,6 ± 10,1 kDa; con estequiometria rK346-HRPc de 1:2; 2:1; 3:1; y 2:2. La conjugación usando sulfo-SMCC se llevó a cabo primero introduciendo grupos -SH en la HRPc usando el reactivo SATA; el antígeno se modificó con sulfo-SMCC. La separación y el análisis de los conjugados se realizaron de forma similar que con el glutaraldehido, resultando en una población heterogénea de conjugados rK346-HRPc con un rango de pesos moleculares entre 150,5 ± 22,6 a 80,0 ± 12,0 kDa; con estequiometria rK346-HRPC de 2:1; 1:2; 2:2 y 1:3, y con una eficiencia de conjugación incrementada en comparación con glutaraldehido. De esta forma, se habilitó al sulfo-SMCC como un reactivo potencial para acoplar antígenos a la HRPc, como método para el diseño de un sistema reportero económico, especifico y fácil de aplicar, útil en la evaluación de individuos en riesgo y/o en estados tempranos o avanzados de leishmaniasis visceral.

Mimotopes for Api g 5, a Relevant Cross-reactive Allergen, in the Celery-Mugwort-Birch-Spice Syndrome

PURPOSE: In the celery-mugwort-birch-spice syndrome, a significant proportion of IgE is directed against high molecular weight (HMW) glycoproteins, including the celery allergen Api g 5. BIP3, a monoclonal antibody originally raised against birch pollen, recognizes HMW allergens in birch and mugwort pollens, celery, and Apiaceae spices. Our aim was to generate mimotopes using BIP3 for immunization against the HMW allergens relevant in the celery-mugwort-birch-spice cross reactivity syndrome. METHODS: Mimotopes were selected from a random-peptide display library by BIP3 and applied in IgE inhibition assays. The 3 phage clones with the highest inhibitory capacity were chosen for immunization of BALB/c mice. Mouse immune sera were tested for IgG binding to blotted birch pollen extract and used for inhibiting patients' IgE binding. Furthermore, sera were tested for binding to Api g 5, to horseradish peroxidase (HRP) as a second glycoprotein, or to non-glycosylated control allergen Phl p 5 in ELISA, and the specific Api g 5-specific IgG titers were determined. RESULTS: Three rounds of biopanning resulted in phage clones exhibiting 7 different sequences including 1 dominant, 1-6-cyclo-CHKLRCDKAIA. Three phage clones had the capacity to inhibit human IgE binding and induced IgG to the HMW antigen when used for immunizing BALB/c mice. The induced BIP3-mimotope IgG reached titers of 1:500 specifically to Api g 5, but hardly reacted to glycoprotein HRP, revealing a minor role of carbohydrates in their epitope. CONCLUSIONS: The mimotopes characterized in this study mimic the epitope of BIP3 relevant for Api g 5, one of the cross-reactive HMW allergens relevant in the celery-mugwort-birch-spice syndrome. BIP3 mimotopes may be used in the future for hyposensitization in this clinical syndrome by virtue of good and specific immunogenicity.

Quantitative Ultrastructural Analysis of Endings Presynaptic to the Tooth Pulp Afferent Terminals in the Trigeminal Oral Nucleus

The ultrastructural parameters related to synaptic release of endings which are presynaptic to tooth pulp afferent terminals (p-endings) were analyzed to understand the underlying mechanism for presynaptic modulation of tooth pulp afferents. Tooth pulp afferents were labelled by applying wheat-germ agglutinin conjugated horseradish peroxidase to the rat right lower incisor, whereafter electron microscopic morphometric analysis with serial section and reconstruction of p-endings in the trigeminal oral nucleus was performed. The results obtained from 15 p-endings presynaptic to 11 labeled tooth pulp afferent terminals were as follows. P-endings contained pleomorphic vesicles and made symmetrical synaptic contacts with labeled terminals. The p-endings showed small synaptic release-related ultrastructural parameters: volume, 0.82 ± 0.45 µm³ (mean ± SD); surface area, 4.50 ± 1.76 µm²; mitochondrial volume, 0.15 ± 0.07 µm³; total apposed surface area, 0.69 ± 0.24 µm²; active zone area, 0.10 ± 0.04 µm²; total vesicle number, 1045 ± 668.86; and vesicle density, 1677 ± 684/µm². The volume of the p-endings showed strong positive correlation with the following parameters: surface area (r=0.97, P<0.01), mitochondrial volume (r=0.56, P<0.05), and total vesicle number (r=0.73, P<0.05). However, the volume of p-endings did not positively correlate or was very weakly correlated with the apposed surface area (r=-0.12, P=0.675) and active zone area (r=0.46, P=0.084). These results show that some synaptic release-related ultrastructural parameters of p-endings on the tooth pulp afferent terminals follow the "size principle" of Pierce and Mendell (1993) in the trigeminal nucleus oralis, but other parameters do not. Our findings may demonstrate a characteristic feature of synaptic release associated with p-endings.

Morphological Analysis of the Myelinated Parent Axons that Innervate Rat Upper Molar Pulps in the Trigeminal Ganglion

Previous studies suggested that myelinated axons innervating rat molar pulps undergo morphological changes in their peripheral course. However, little information is available on the morphological feature of the parent axons at the site of origin. We therefore investigated the size of the myelinated parent axons and their morphological features at the proximal sensory root of the trigeminal ganglion by horseradish peroxidase (HRP) injection into rat upper molar pulps and subsequent light and electron microscopy. A total of 248 HRP-labeled myelinated axons investigated were highly variable in the size. Fiber area, fiber diameter, axon area (axoplasm area), axon diameter (axoplasm diameter), and myelin thickness were 11.32 +/- 8.36 microm2 (0.80~53.17 microm2), 3.99 +/- 1.53 microm (1.08~9.26 microm), 8.70 +/- 6.30 microm2 (0.70~41.83 microm2), 3.13 +/- 1.13 microm (0.94~7.20 microm) and 0.43 +/- 0.23 microm (0.07~1.06 microm), respectively. The g-ratio (axon diameter / fiber diameter) of the labeled axons was 0.79 +/- 0.05 (0.61~0.91). Axon diameter was highly correlated with myelin thickness (correlation coefficients,r=0.83) but little correlated with g-ratio (r=-0.33) of individual myelinated parent axons. These results indicate that myelin thickness of the myelinated parent axons innervating rat molar pulps increase with increasing axon diameter, thus maintaining a constant g-ratio.

Kinetics of enzymatic modification of quercetin with cysteine by horseradish peroxidase.

The kinetic mechanism of enzymatic modification of flavonol quercetin with L-cysteine by horseradish peroxidase (HRP) was studied. Reaction of modification of quercetin was followed by recording spectral changes over time at 380 nm. All reactions were performed in 100 mM phosphate buffer pH, 6.0 at 20ºC. Kinetic parameters were determined from graphics of linear Michaelis-Menten equation. The values obtained at specified intervals were: Vmax = 0.17 ÷ 0.91 ΔA380/min, Km = 0.023 ÷ 0.5 mM, kcat = 0.21 ÷ 1.14 ΔA380/min∙nM-1 and Vmax/Km = 0.83 ÷ 26.55 ΔA380/min∙mM-1. It was found that all investigated reactions of the modification of quercetin with L-cysteine by HRP followed an ordered mechanism. We propose that HRP initially reacts with H2O2 than with quercetin and finally with L-cysteine, leading to the introduction of L-cysteine in the structure of quercetin.

Mucosal Mast Cell Count Is Associated With Intestinal Permeability in Patients With Diarrhea Predominant Irritable Bowel Syndrome

BACKGROUND/AIMS: Although mucosal mast cell tryptase is known to significantly increase intestinal permeability, the relationship between mucosal mast cells and intestinal permeability remains unclear. The objective of this study was to evaluate the correlation among intestinal permeability, tryptase activity and mucosal mast cell count. METHODS: Rectal biopsies from 16 patients with diarrhea-predominant irritable bowel syndrome (IBS-D) and 7 normal subjects were assessed for tryptase activity and macromolecular permeability using horseradish peroxidase in Ussing chambers. In addition, mucosal mast cell levels were immunohistochemically quantified via image analysis. RESULTS: Rectal biopsy of tissues from IBS-D patients showed significantly increased permeability compared with those from normal controls (0.644 +/- 0.08 and 0.06 +/- 0.00 ng/2 hr/mm2, P 0.05). However, correlation analysis revealed that only mucosal mast cell count was significantly correlated with intestinal permeability in IBS-D patients (r = 0.558, P < 0.05). CONCLUSIONS: This study demonstrated a positive correlation between the number of mucosal mast cells and intestinal permeability, suggesting that mucosal mast cells play an important role for increased intestinal permeability in patients with IBS-D.

Efecto de la peroxidasa sobre la resistencia de unión de una resina compuesta al esmalte dental posblanqueamiento / Effect of peroxidase on composite resin adhesion to dental enamel after whitening

Introducción: se ha reportado que el oxígeno residual liberado por los agentes blanqueadores interfieren en la adhesión de las resinas compuestas a la estructura dental, razón por la cual se tiene como objetivo comparar la resistencia de unión al corte (RUC) de una resina compuesta al esmalte dental posblanqueamiento con peróxido de hidrógeno al 38%, antes y después de tratar la superficie con la enzima peroxidasa previo a la adhesión. Métodos: se seleccionaron 45 premolares humanos sanos, divididos entres grupos de 15 dientes cada uno. Grupo 1: control (solo adhesión); grupo 2: blanqueamiento y adhesión; grupo 3: blanqueamiento, aplicación de peroxidasa y adhesión. Posterior al tratamiento se midió la RUC en la máquina de ensayos Shimadzu, para determinar diferencia estadísticamente significativa entre los tres grupos con nivel de confianza de 95% y con valor de p < 0,05. Resultados: el grupo control obtuvo la RUC de 12,8 Mpa (± 3,2), el grupo con blanqueamiento tuvo el promedio de 3,5 Mpa (± 1,43) y el grupo con blanqueamiento y aplicación de peroxidasa presentó promedio de 12,2 Mpa (± 3,12). Conclusiones: los valores de RUC disminuyeronsignificativamente con la aplicación de peróxido de hidrógeno al 38%, sin embargo, se logró el aumento significativo al aplicar la peroxidasa previa a la adhesión.

Introduction: some reports suggest that residual oxygen released by whitening agents interfere with composite resinadhesion to dental structure. The objective of this study is therefore to compare composite resins’ shear bond strength to dental enamel after whitening using 38% hydrogen peroxide pre- and post- treatment with peroxidase enzyme before adhesion. Methods: a total of 45 healthy human premolars were selected and divided into three groups of 15 teeth each. Group 1: control group (only adhesion); group 2: whitening and adhesion; group 3: whitening, application of peroxidase, and adhesion. After treatment, shear bond strength was measured with a Shimadzu testing machine in order to determine significant statistical differences among the three groups, with a confidence level of 95% and p < 0.05. Results: the control group obtained a shear bond strength of 12.8Mpa (± 3.2), the whitening group showed an average 3.5 Mpa (± 1.43), and the group treated with whitening and peroxidase presented an average 12.2 Mpa (± 3.12). Conclusions: shear bond strength values significantly decreased with application of 38% hydrogen peroxide; however, a significant increase was also obtained byapplying peroxidase before adhesion.

Anti-tumor effect of suicide gene therapy using chimeric promoter plus radiotherapy on cancer cell lines / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To explore the synergistic anti-tumor effect of radiotherapy and horseradish peroxidase/prodrug indole-3-acetic acid (HRP/IAA) gene therapy system using chimeric hTERT promoter responsive to ionizing radiation.</p><p><b>METHODS</b>The synthetic hTERT promoters containing four tandem-repeat copies of radio-inducible CArG elements, and the chimeric promoter containing cytomegalovirus (CMV) early promoter were both constructed. The activities of the chimeric promoters in cancer cell lines (HeLa, A549, and MHCC97) and normal cell line (MRC-5) were detected using luciferase reporter gene expression analysis after a (60)Co γ-irradiation treatment at a series of doses(a single dose of 0 to 10 Gy). The anti-tumor effect of combining irradiation with HRP/IAA gene-directed enzyme prodrug therapy system controlled by the chimeric promoter was tested by colony formation assay, cell counting and apoptosis analysis.</p><p><b>RESULTS</b>The chimeric promoters were ineffective in normal human cells, even after irradiation, but the expression of luciferase gene in tumor cells was significantly higher. The activity of the chimeric promoter in MRC-5 cells was 22.3%, 12.9% and 13.6% of that in HeLa, A549 and MHCC97 cells, respectively. After irradiation, the ratios were 11.7%, 8.7% and 8.8%, respectively. Furthermore, the chimeric promoters could successfully induce the expression of luciferase gene following different doses of radiation, with maximal inducible activity seen after 6 Gy irradiation. The chimeric promoter containing four tandem-repeat copies of radio-inducible CArG elements and CMV early promoter showed the highest activity with 6 Gy irradiation. The relative luciferase activities in HeLa, A549 and MHCC97 cells were 1.7 ± 0.2, 2.3 ± 0.2 and 2.3 ± 0.1, respectively. The chimeric promoter mediated suicide gene therapy system could increase radio-sensitivity in different cancer cells. Compared with the control system, it plus irradiation showed stronger cell proliferation inhibition, 67.3% vs. 26.1% in HeLa, 69.0% vs. 28.3% in A549, 64.6% vs. 20.8% in MHCC97 cells, and also higher apoptosis-inducing effect, 39.6% vs. 14.2% in HeLa, 33.0% vs. 12.4% in A549, and 33.2% vs. 14.2% in MHCC97 cells.</p><p><b>CONCLUSIONS</b>Chimeric promoter containing hTERT promoter, CArG elements and CMV promoter preserve the tumor-specificity in telomerase-positive tumor cells, and irradiation-responsive to low dose of radiation. The suicide gene therapy using this promoter plus radiotherapy show a strong anti-tumor effect in vitro. It is expected to have a good potential for future application in gene radiotherapy.</p>

Lectin histochemical study of vasculogenesis during rat pituitary morphogenesis

The aim of this study was to investigate glycoconjugates distribution patterns as well as their changes during the course of pituitary portal vasculogenesis and angiogenesis. Formalin fixed paraffin sections of 10 to 20 days of Sprague Dawly rat fetuses were processed for histochemical studies using four different horseradish peroxidase [HRP] conjugated lectins. Orange peel fungus [OFA], Vicica villosa [VVA], Glycine max [SBA] and Wistaria floribunda [WFA] specific for alpha-L-Fucose, D-Gal, alpha, beta-D-GalNAc and D-GalNAc terminal sugars of glycoconjugates respectively. Our finding indicated that adenohypophysal cells reacted with OFA on gestational day 10 E[10] and increased progressively to E[14]. Staining intensity did not change from days 14 to17, then after increased following days to E[20] significantly [P< 0.05]. A few cells around Rathke's pouch reacted with VVA on E[13], increased to E[14] and decreased significantly afterward [P< 0.05]. Reaction of some cells around Rathke's pouch reacted with SBA on E[14]. This visible reaction was the same as E[18] and decreased later [P< 0.05]. Many cells around Rathke's pouch reacted with WFA on E[13] and increased on E[14] and E[15] and decreased thereafter [P<0.05]. Reactions of OFA and other tested lectins with endothelial cells around Rathke's pouch and developing pars distalis were different. These results suggest that embryonic origin of hypophiseal pituitary portal [HPP] system endothelial cells are not the same and our finding also indicated that glycoconjugates with terminal sugars alpha-L-Fucose, D-Gal, alpha, beta-D-GalNAc may play critical role[s] in cell interactions and tissue differentiations such as vasculogensis and angiogenesis as well as other developmental precursors in formation of the pituitary gland

Matrix metalloproteinase-1 expression in oral submucous fibrosis: An immunohistochemical study.

Context: Oral submucous fibrosis (OSF) is a form of pathological fibrosis affecting the oral mucosa. There is compelling evidence to implicate the habitual chewing of areca nut with the development of OSF. Because collagens are the major structural components of connective tissues, including oral submucosa, the composition of collagen within each tissue needs to be precisely regulated to maintain tissue integrity. Arecoline stimulates fibroblasts to increase the production of collagen by 150%. Aim: As the role of collagenase is implicated in cleaving the collagen under physical conditions, this study was carried out to evaluate the role of collagenase-1 (matrix metalloproteinase [MMP]-1) in a pathologic condition like OSF. Settings and Design: A total of 40 patients were included in the study, comprising of 30 OSF as Group 1 and 10 normal buccal mucosa tissue as Group 2. Materials and Methods: Both the groups were stained for MMP-1 by the immunohistochemical method using the streptavidin HRP-biotin labeling technique. MMP-1 expression intensity in the epithelium and connective tissue was decreased in Group 1 when compared to Group 2. Statistical Analysis Used: Chi-square test of association was used to determine the difference in the expression of MMP-1 between OSF and normal buccal mucosa and among different histological gradings of OSF. Results: The results were statistically significant. However, there was no statistically significant difference between the expression of MMP-1 among different histological grades of OSF in Group 1.

The triad Indole-3-Acetic Acid Ethyl Ester/Esterase/Horseradish Peroxidase as a new cytotoxic Prodrug/Enzyme combination

The antibody-directed enzyme prodrug therapy (ADEPT) is a means of restricting the action of toxic drugs to the tumor site. The enzyme/prodrug pair horseradish peroxidase (HRP)/indole-3-acetic acid (IAA) has been studied as a combination with potential application in ADEPT strategies. In this combination, the non-toxic plant hormone IAA is activated to cytotoxic species by the catalytic action of HRP. Objective: We studied the use of the ethyl ester of IAA as a new prodrug that could be activated by two enzymes, HRP and esterase. Methods: The oxidation of IAA and its ethyl ester, catalyzed by HRP, was monitored by the consumption of dioxygen and liquid chromatography. The cytotoxicity of IAA and its ethyl ester in combination with HRP and esterase was assessed using the lineage McCoy cells through the trypan blue and neutral red assays. Results: We found that HRP was not able to catalyze the oxidation of IAA-ethyl ester in the absence of an additional esterase. Hence, the potential cytotoxicity of the IAA-ethyl ester could be controlled by sequential treatment with esterase, to liberate the carboxyl group, and HRP, for oxidation and generation of cytotoxic species. We present evidence for the potential application of the combination IAA-ethyl ester/esterase/horseradish peroxidase as a new ADEPT, GDEPT or related strategy. Conclusions: We suggest that this technique could provide more selectivity in the generation of cytotoxic drugs at tumor sites.

Immortalized mouse brain endothelial cell line Bend.3 displays the comparative barrier characteristics as the primary brain microvascular endothelial cells / 中国当代儿科杂志

<p><b>OBJECTIVE</b>The purpose of this study was to assess weather the immortalized mouse brain endothelial cell line Bend.3 displays the comparative barrier characteristics as the primary brain microvascular endothelial cells (BEMC).</p><p><b>METHODS</b>Immortalized mouse brain endothelial cell line, Bend.3 cells were cultured in transwell inserts and their restrictive characteristics were assessed by transendothelial electrical resistance (TEER) and horseradish peroxidase (HRP) permeability assays. Western blot and direct fluorescent staining methods were used to detect the tight junction protein expression and F-actin distribution.</p><p><b>RESULTS</b>The TEER in Bend.3 cells increased with the prolonged culture time and increased to 82.3+/-6.0 Omega cm2 10 days after culture, which was significantly higher than that 3 days after culture (37.3+/-3.1 Omega cm2; P<0.05). There were significant differences in the permeability rates for HRP 3 and 10 days after culture (4.3+/-0.20)% vs (2.2+/-0.05)% (P<0.05). Western blot indicated high level expression of tight junction proteins occludin and ZO-1 in Bend.3 cells 10 days after culture. F-actin was visualized around the cell membrane and presented scrobiculate linear fluorescence 10 days after culture.</p><p><b>CONCLUSIONS</b>Bend.3 cells have similar barrier characteristics to BEMC, and their barrier function may reach to the best effect 10 days after culture.</p>

Stabilizers of horseradish peroxidase / 生物工程学报

Keeping an enzyme in its native form with high catalytic activity is of great significance. In the present study, thermal stabilizers of horseradish peroxidase (HRP) were screened. The results indicated that thermal stability of HRP was enhanced by magnesium sulphate and gelatin. A synergic effect of magnesium sulphate and gelatin was observed. In the presence of the stabilizer, the enzymatic activity of HRP remained 89% after kept for 80 h at 50 degrees C and 57% for 90 days at room temperature. Thermal alterations of HRP structure in the absence and presence of the stabilizers were explored by using UV absorption spectra at 402 nm (Soret band), intrinsic fluorescence and 8-anilinonaphthalene-1-sulfonic acid (ANS) fluorescence. The results suggested that magnesium sulphate and gelatin attenuated the extent of unfolding of HRP and therefore the native enzyme structure was stabilized.

Optimized Immunohistochemical Analysis of Cerebellar Purkinje Cells Using a Specific Biomarker, Calbindin D28k

Cerebellar Purkinje cells (PCs) play a crucial role in motor functions and their progressive degeneration is closely associated with spinocerebellar ataxias. Although immunohistochemical (IHC) analysis can provide a valuable tool for understanding the pathophysiology of PC disorders, the method validation of IHC analysis with cerebellar tissue specimens is unclear. Here we present an optimized and validated IHC method using antibodies to calbindin D28k, a specific PC marker in the cerebellum. To achieve the desired sensitivity, specificity, and reproducibility, we modified IHC analysis procedures for cerebellar tissues. We found that the sensitivity of staining varies depending on the commercial source of primary antibody. In addition, we showed that a biotin-free signal amplification method using a horseradish peroxidase polymer-conjugated secondary antibody increases both the sensitivity and specificity of ICH analysis. Furthermore, we demonstrated that dye filtration using a 0.22 micrometer filter eliminates or minimizes nonspecific staining while preserving the analytical sensitivity. These results suggest that our protocol can be adapted for future investigations aiming to understand the pathophysiology of cerebellar PC disorders and to evaluate the efficacy of therapeutic strategies for treating these diseases.