Strictinin, a Major Ingredient in Yunnan Kucha Tea Possessing Inhibitory Activity on the Infection of Mouse Hepatitis Virus to Mouse L Cells.

Theacrine and strictinin of Yunnan Kucha tea prepared from a mutant variety of wild Pu'er tea plants were two major ingredients responsible for the anti-influenza activity. As the COVID-19 outbreak is still lurking, developing safe and cost-effective therapeutics is an urgent need. This study aimed to evaluate the effects of these tea compounds on the infection of mouse hepatitis virus (MHV), a ß-coronavirus serving as a surrogate for SARS-CoV. Treatment with strictinin (100 µM), but not theacrine, completely eliminated MHV infection, as indicated by a pronounced reduction in plaque formation, nucleocapsid protein expression, and progeny production of MHV. Subsequently, a time-of-drug addition protocol, including pre-, co-, or post-treatment, was exploited to further evaluate the possible mechanism of antiviral activity mediated by strictinin, and remdesivir, a potential drug for the treatment of SARS-CoV-2, was used as a positive control against MHV infection. The results showed that all three treatments of remdesivir (20 µM) completely blocked MHV infection. In contrast, no significant effect on MHV infection was observed when cells were pre-treated with strictinin (100 µM) prior to infection, while significant inhibition of MHV infection was observed when strictinin was introduced upon viral adsorption (co-treatment) and after viral entry (post-treatment). Of note, as compared with the co-treatment group, the inhibitory effect of strictinin was more striking in the post-treatment group. These results indicate that strictinin suppresses MHV infection by multiple mechanisms; it possibly interferes with viral entry and also critical step(s) of viral infection. Evidently, strictinin significantly inhibited MHV infection and might be a suitable ingredient for protection against coronavirus infection.

Electrospun cellulose acetate/gelatin nanofibrous wound dressing containing berberine for diabetic foot ulcer healing: in vitro and in vivo studies.

Functional wound dressing with tailored physicochemical and biological properties is vital for diabetic foot ulcer (DFU) treatment. Our main objective in the current study was to fabricate Cellulose Acetate/Gelatin (CA/Gel) electrospun mat loaded with berberine (Beri) as the DFU-specific wound dressing. The wound healing efficacy of the fabricated dressings was evaluated in streptozotocin-induced diabetic rats. The results demonstrated an average nanofiber diameter of 502 ± 150 nm, and the tensile strength, contact angle, porosity, water vapor permeability and water uptake ratio of CA/Gel nanofibers were around 2.83 ± 0.08 MPa, 58.07 ± 2.35°, 78.17 ± 1.04%, 11.23 ± 1.05 mg/cm2/hr, and 12.78 ± 0.32%, respectively, while these values for CA/Gel/Beri nanofibers were 2.69 ± 0.05 MPa, 56.93 ± 1°, 76.17 ± 0.76%, 10.17 ± 0.21 mg/cm2/hr, and 14.37 ± 0.42%, respectively. The antibacterial evaluations demonstrated that the dressings exhibited potent antibacterial activity. The collagen density of 88.8 ± 6.7% and the angiogenesis score of 19.8 ± 3.8 obtained in the animal studies indicate a proper wound healing. These findings implied that the incorporation of berberine did not compromise the physical properties of dressing, while improving the biological activities. In conclusion, our results indicated that the prepared mat is a proper wound dressing for DFU management and treatment.

Controlling the Release of Proteins from Therapeutic Nanofibers: The Effect of Fabrication Modalities on Biocompatibility and Antimicrobial Activity of Lysozyme.

Therapeutic application of pharmacologically active proteins requires advanced drug delivery systems for stabilizing their activity and preventing denaturation during storage and patient treatment. Depending on their clinical target, controlled drug release is often required to achieve the intended therapeutic effect. In this context, electrospun nanofibers gained considerable attention. However, even though immediate drug release from such fibers can easily be realized, fiber mat fabrication providing long-term controlled protein release still bares challenges.In this study, lysozyme was encapsulated in poly(vinyl alcohol) fibers followed by post-modification with MeOH, glutaraldehyde vapor, or UV light. Subsequently, a systematic investigation of the effect of these post-modification treatments on the physicochemical properties of the fibers and the stability and release kinetics of lysozyme was performed. MeOH treatment did not affect lysozyme release kinetics compared to untreated fibers, whereas glutaraldehyde vapor and UV light treatment prolonged the drug release. Infrared spectroscopy revealed cross-linking of the polymer by glutaraldehyde vapor, which reduced the lysozyme release from the fibers. Further, protein activity was significantly reduced for fibers treated with glutaraldehyde vapor and UV light. In addition, reduced viability was identified for cells in contact with glutaraldehyde vapor-treated fibers and, to a lesser extent, for UV light-treated fibers, whereas MeOH-treated fibers did not affect cell viability. These results elucidated the effects of fiber post-modification on the release kinetics, activity, and biocompatibility of protein drugs and can serve as guidance for rational development of nanomedicines for safe and effective therapeutic delivery of natural proteins.

Angelica dahurica Extracts Improve Glucose Tolerance through the Activation of GPR119.

G protein-coupled receptor (GPR) 119 is expressed in pancreatic ß-cells and intestinal L cells, and is involved in glucose-stimulated insulin secretion and glucagon-like peptide-1 (GLP-1) release, respectively. Therefore, the development of GPR119 agonists is a potential treatment for type 2 diabetes. We screened 1500 natural plant extracts for GPR119 agonistic actions and investigated the most promising extract, that from Angelica dahurica (AD), for hypoglycemic actions in vitro and in vivo. Human GPR119 activation was measured in GeneBLAzer T-Rex GPR119-CRE-bla CHO-K1 cells; intracellular cAMP levels and insulin secretion were measured in INS-1 cells; and GLP-1 release was measured in GLUTag cells. Glucose tolerance tests and serum plasma insulin levels were measured in normal C57BL6 mice and diabetic db/db mice. AD extract-treated cells showed significant increases in GPR119 activation, intracellular cAMP levels, GLP-1 levels and glucose-stimulated insulin secretion as compared with controls. In normal mice, a single treatment with AD extract improved glucose tolerance and increased insulin secretion. Treatment with multiple doses of AD extract or n-hexane fraction improved glucose tolerance in diabetic db/db mice. Imperatorin, phellopterin and isoimperatorin were identified in the active fraction of AD extract. Among these, phellopterin activated GPR119 and increased active GLP-1 and insulin secretion in vitro and enhanced glucose tolerance in normal and db/db mice. We suggest that phellopterin might have a therapeutic potential for the treatment of type 2 diabetes.

Antifungal Activity and Action Mechanism of Histatin 5-Halocidin Hybrid Peptides against Candida ssp.

The candidacidal activity of histatin 5 is initiated through cell wall binding, followed by translocation and intracellular targeting, while the halocidin peptide exerts its activity by attacking the Candida cell membrane. To improve antimicrobial activities and to understand the killing mechanism of two peptides, six hybrid peptides were designed by conjugating histatin 5 and halocidin. A comparative approach was established to study the activity, salt tolerance, cell wall glucan binding assay, cytotoxicity, generation of ROS and killing kinetics. CD spectrometry was conducted to evaluate secondary structures of these hybrid peptides. Furthermore the cellular localization of hybrid peptides was investigated by confocal fluorescence microscopy. Of the six hybrid congeners, di-PH2, di-WP2 and HHP1 had stronger activities than other hybrid peptides against all tested Candida strains. The MIC values of these peptides were 1-2, 2-4 and 2-4 µg/ml, respectively. Moreover, none of the hybrid peptides was cytotoxic in the hemolytic assay and cell-based cytotoxicity assay. Confocal laser microscopy showed that di-PH2 and HHP1 were translocated into cytoplasm whereas di-WP2 was accumulated on surface of C. albicans to exert their candidacidal activity. All translocated peptides (Hst 5, P113, di-PH2) were capable of generating intracellular ROS except HHP1. Additionally, the KFH residues at C-terminal end of these peptides were assumed for core sequence for active translocation.

Dual properties of hispidulin: antiproliferative effects on HepG2 cancer cells and selective inhibition of ABCG2 transport activity.

Hepatocellular carcinoma is the third most common cause of cancer-related deaths worldwide. Furthermore, the existing pharmacological-based treatments are insufficiently effective and generate many side effects. Hispidulin (6-methoxy-5,7,4'-trihydroxyflavone) is a flavonoid found in various medicinal herbs that present antineoplastic properties. Here we evaluated how modulation of reactive oxygen species (ROS) and alterations of antioxidant defenses could be associated to the antiproliferative effects of hispidulin in HepG2 cells. In addition, we studied the inhibitory activity of hispidulin on the efflux of drugs mediated by ABC transporters involved in multidrug resistance. In order to understand the increase of intracellular ROS promoted by hispidulin, we investigated the mRNA expression levels and activities of antioxidant enzymes, and the GSH/GSSG ratio. We showed that hispidulin significantly down-regulated the transcription levels of catalase, leading to reduction of enzyme activity and decrease of the GSH content. We also observed that, in the presence of N-acetylcysteine or exogenous catalase, the proliferation was lowered back to the control levels. These data clearly indicate a strong involvement of intracellular ROS levels for triggering the antiproliferative effects. We also demonstrated that the inhibition produced by hispidulin on drug efflux was specific for ABCG2, since no effects were observed with ABCB1 and ABCC1. Furthermore, HepG2 cells were more sensitive to hispidulin-mediated cell death than immortalized L929 fibroblasts, suggesting a differential toxicity of this compound between tumor and non-tumor cell lines. Our results suggest that hispidulin constitutes a promising candidate to sensitize chemoresistant cancer cells overexpressing ABCG2.

Phototoxic effect of curcumin on methicillin-resistant Staphylococcus aureus and L929 fibroblasts.

Photodynamic therapy has been investigated as an alternative method of killing pathogens in response to the multiantibiotic resistance problem. This study evaluated the photodynamic effect of curcumin on methicillin-resistant Staphylococcus aureus (MRSA) compared to susceptible S. aureus (MSSA) and L929 fibroblasts. Suspensions of MSSA and MRSA were treated with different concentrations of curcumin and exposed to light-emitting diode (LED). Serial dilutions were obtained from each sample, and colony counts were quantified. For fibroblasts, the cell viability subsequent to the curcumin-mediated photodynamic therapy was evaluated using the MTT assay and morphological changes were assessed by SEM analysis. Curcumin concentrations ranging from 5.0 to 20.0 µM in combination with any tested LED fluences resulted in photokilling of MSSA. However, only the 20.0 µM concentration in combination with highest fluence resulted in photokilling of MRSA. This combination also promoted an 80% reduction in fibroblast cell metabolism and morphological changes were present, indicating that cell membrane was the main target of this phototherapy. The combination of curcumin with LED light caused photokilling of both S. aureus strains and may represent an alternative treatment for eradicating MRSA, responsible for significantly higher morbidity and mortality and increased healthcare costs in institutions and hospitals.

Murine Borrelia arthritis is highly dependent on ASC and caspase-1, but independent of NLRP3.

INTRODUCTION: The protein platform called the NOD-like-receptor -family member (NLRP)-3 inflammasome needs to be activated to process intracellular caspase-1. Active caspase-1 is able to cleave pro-Interleukin (IL)-1ß, resulting in bioactive IL-1ß. IL-1ß is a potent proinflammatory cytokine, and thought to play a key role in the pathogenesis of Lyme arthritis, a common manifestation of Borrelia burgdorferi infection. The precise pathways through which B. burgdorferi recognition leads to inflammasome activation and processing of IL-1ß in Lyme arthritis has not been elucidated. In the present study, we investigated the contribution of several pattern recognition receptors and inflammasome components in a novel murine model of Lyme arthritis. METHODS: Lyme arthritis was elicited by live B. burgdorferi, injected intra-articularly in knee joints of mice. To identify the relevant pathway components, the model was applied to wild-type, NLRP3-/-, ASC-/-, caspase-1-/-, NOD1-/-, NOD2-/-, and RICK-/- mice. As a control, TLR2-/-, Myd88-/- and IL-1R-/- mice were used. Peritoneal macrophages and bone marrow-derived macrophages were used for in vitro cytokine production and inflammasome activation studies. Joint inflammation was analyzed in synovial specimens and whole knee joints. Mann-Whitney U tests were used to detect statistical differences. RESULTS: We demonstrate that ASC/caspase-1-driven IL-1ß is crucial for induction of B. burgdorferi-induced murine Lyme arthritis. In addition, we show that B. burgdorferi-induced murine Lyme arthritis is less dependent on NOD1/NOD2/RICK pathways while the TLR2-MyD88 pathway is crucial. CONCLUSIONS: Murine Lyme arthritis is strongly dependent on IL-1 production, and B. burgdorferi induces inflammasome-mediated caspase-1 activation. Next to that, murine Lyme arthritis is ASC- and caspase-1-dependent, but NLRP3, NOD1, NOD2, and RICK independent. Also, caspase-1 activation by B. burgdorferi is dependent on TLR2 and MyD88. Based on present results indicating that IL-1 is one of the major mediators in Lyme arthritis, there is a rationale to propose that neutralizing IL-1 activity may also have beneficial effects in chronic Lyme arthritis.

Antiviral activity of Bignoniaceae species occurring in the State of Minas Gerais (Brazil): part 1.

AIMS: To evaluate the antiviral activity of Bignoniaceae species occurring in the state of Minas Gerais, Brazil. METHODS AND RESULTS: Ethanol extracts of different anatomical parts of bignoniaceous plant species have been evaluated in vitro against human herpesvirus type 1 (HSV-1), vaccinia virus (VACV) and murine encephalomyocarditis virus (EMCV) by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. A total of 34 extracts from 18 plant species selected according to ethnopharmacological and taxonomic criteria were screened. Fifteen of the 34 extracts (44.1%) have disclosed antiviral activity against one or more of the viruses assayed with EC(50) values in the range of 23.2 ± 2.5-422.7 ± 10.9 µg ml(-1). CONCLUSIONS: Twelve of the 34 extracts (35.3%) might be considered promising sources of antiviral natural products, as they have shown EC50 ≤ 100 µg ml(-1). The present screening discloses the high potential of the Bignoniaceae family as source of antiviral agents. SIGNIFICANCE AND IMPACT OF THE STUDY: Active extracts were identified and deserve bioguided studies for the isolation of antiviral compounds and studies on mechanism of action.

The effects of low-level diode laser treatment and dental pulp-capping materials on the proliferation of L-929 fibroblasts.

Low-level laser therapy (LLLT) has been reported to improve tissue healing and might therefore be useful in dental pulp capping after trauma. We evaluated the effects of a low-level diode laser (lambda = 680 nm) and dental pulp-capping substances on cell proliferation. Calcium hydroxide and adhesive resin were applied as conditioned media to cultures. Half of the samples received irradiation with the diode laser at a fluence of 4 J/cm(2) for 60 s. Using a hemocytometer, cells were counted at 1, 3, 5, and 7 days, and the data were analyzed by ANOVA. All cultures exhibited continuous growth, except those treated with adhesive resin. As compared to the other two groups, cell proliferation was significantly lower in cultures treated with adhesive resin; it was also significantly lower in cultures treated with calcium hydroxide, as compared to the control group. When combined with dental pulp-capping materials, LLLT had no effect on L-929 cell proliferation.

Actividad citotóxica de physalis peruviana (aguaymanto) en cultivos celulares de adenocarcinoma colorectal, próstata y leucemia mieloide crónica / Cytotoxic effect of physalis peruviana in cell culture of colorectal and prostate cancer and chronic myeloid leukemia

INTRODUCCIÓN: Las plantas han sido empleadas como drogas por siglos. Sin embargo, se ha investigado poco sobre su gran potencial como fuentes de nuevos agentes terapéuticos. El objetivo del presente trabajo fue evaluar la actividad citotóxica de los extractos etanólicos de tallos y hojas de Physalis peruviana, sobre las líneas celulares HT-29, PC-3, K-562 y VERO. MATERIALES Y MÉTODOS: Las líneas celulares HT-29, PC-3, K-562 y VERO, fueron expuestas a cuatro concentraciones de extractos etanólicos de tallos y hojas de Physalis peruviana. Asimismo, a diferentes concentraciones de cisplatino y 5-Fluorouracilo (5-FU), que se usaron como controles positivos. Se hallaron los porcentajes de crecimiento en 48 horas, luego se determinó la concentración inhibitoria 50 (IC50) mediante análisis de regresión lineal y el índice de selectividad de cada muestra. RESULTADOS: Los extractos etanólicos de tallos y hojas de Physalis peruviana mostraron actividad citotóxica: Los CI50 en μg/mL en hojas y tallos fueron, 0.35 (r=-0.95 p<0.025) y 0.37 (r=-0,90 p<0.05) para HT-29; 0.87 (r=-0.98 p<0.01) y 1.01 (r=-0.95 p<0.025) para PC-3; 0.02 (r=-0.98 p<0.01) y 0.03 (r=-0.98 p<0.01) para K-562; 4.9 (r=-0.95 p<0.025) y 6.2 (r=-0.98 p<0.01) para VERO. Los CI50 para los antineoplásicos fueron: para el cisplatino: 4.2 (r=-0.96 p<0.025), 10.3 (r=-0.97 p<0.025), 0.15 (r=-0.98 p=0.01), y 1.1 (r=-0.98 p=0.01). Parael 5-FU: 2.3 (r=-0.97 p<0.025), 17.9 (r=-0.95 p<0.025), 0.15 (r=-0.98 p=0.01), y 1.1 (r=-0.94 p=0.05) para HT-29, PC-3, K562 y VERO, respectivamente. Los índices de selectividad de los extractos de tallos y hojas, estuvieron entre 5.6 y 245 para las líneas celulares tumorales evaluadas; por el contrario, el cisplatino y el 5-FU, solo alcanzaron valores entre 0.11 y 7.3...

INTRODUCTION: The plants have been used as drugs for centuries. However, limited research has been done on its great potential as sources of new therapeutic agents. The purpose of this study was to evaluate Physalis peruviana cytotoxic activity on cell lines HT-29, PC-3, K-562 and VERO. Materials and Methods: The HT-29 cell lines, PC-3, K-562 and VERO, were exposed to four concentrations of P. peruviana ethanolic leave and stem extracts, also at different concentrations of cisplatin and 5-fluorouracil (5-FU), which were used as positive controls. We found rates of growth within 48 hours, then we determined the inhibitory concentration 50 (IC50) using linear regression analysis and the index of selectivity of each sample. RESULTS: The P. peruviana ethanolic leave and stem extracts showed cytotoxic activity. The IC50 in μg/mL in leaves and stems were, 0.35 (r =-0.95 p <0.025) and 0.37 (r =- 0.90 p <0.05 ) for HT-29; 0.87 (r =-0.98 p <0.01) and 1.01 (r =-0.95 p <0.025) for PC-3; 0.02 (r =-0.98p <0.01) and 0.03 (r =-0.98 p <0.01) for K-562; 4.9 (r =-0.95 p <0.025) and 6.2 (r =-0.98 p <0.01) for VERO. The IC50 for antineoplastic were: for cisplatin: 4.2 (r =-0.96 p <0.025),10.3 (r =-0.97 p <0.025), 0.15 (r =-0.98 p = 0.01) and 1.1 (r =- 0.98 p = 0.01); for 5-FU: 2.3 (r =-0.97 p <0.025), 17.9 (r =-0.95 p <0.025), 0.15 (r =-0.98 p = 0.01) and 1.1 (r =-0.94 p = 0.05) for HT-29, PC-3, K562 and VERO respectively. The leaves and stems extracts selectivity index were between 5.6 and 245 for tumor cell lines evaluated, by contrast, cisplatin and 5-FU, only showed values between 0.11 and 7.3. CONCLUSIONS: The P. peruviana leaves and steams ethanolic extracts were more cytotoxic than cisplatin and 5 FU, on the lines HT-29, PC-3 and K562. Furthermore the P. peruviana cytotoxic effects were less than cisplatin and 5-FU for VERO control cells lines.

A novel immunoregulatory protein in human colostrum, syntenin-1, for promoting the development of IgA-producing cells from cord blood B cells.

Human colostrum contains many bioactive factors that must promote the development of intestinal mucosal immunity in infants. Especially, the presence of certain cytokines such as transforming growth factor (TGF)-beta or IL-10 has been of great interest for IgA production as a function of mucosal immune response. In the present study, we attempted to investigate whether unidentified factors inducing generation of IgA-producing cells from naive B cells might exist in colostrum. For this purpose, colostrum samples were directly added to a culture consisting of naive B cells and dendritic cells from cord blood and CD40 ligand-transfected L cells, comparing with recombinant IL-10 (rIL-10) and/or rTGF-beta. It was noted that most colostrum samples alone were able to induce IgA-secreting cells at higher levels than rIL-10 and/or rTGF-beta. IgA-inducing activity of colostrum was abolished by neither anti-neutralizing mAbs against IL-10 nor TGF-beta, though partially by anti-IL-6 mAb. We prepared partially purified fractions from both pooled colostrums with and without IgA-inducing activity and comparatively performed quantitative proteomic analysis by two-dimensional difference gel electrophoresis followed by liquid chromatography-mass spectrometry. As a result, syntenin-1 was identified as a candidate for IgA-inducing protein in colostrum. Western blot analysis indicated that levels of syntenin-1 in colostrum samples were correlated with their IgA-inducing activities. Moreover, we demonstrated that recombinant syntenin-1 could induce preferentially IgA production from naive B cells. These results suggest that syntenin-1 serves as one of IgA-inducing factors for B cells.

Sanionins: anti-inflammatory and antibacterial agents with weak cytotoxicity from the Antarctic moss Sanionia georgico-uncinata.

Sanionins A (1) and B (2) were isolated from the moss Sanionia georgico-uncinata, collected on the Antarctic Livingston Island. The compounds 1 and 2 were purified by solvent extraction, silica gel column chromatography, and preparative HPLC, consecutively. The structures of the both compounds were elucidated by 1D and 2D NMR experiments and mass spectrometric investigations. These compounds showed activity against important Gram-positive pathogens, such as mycobacteria, multiresistant staphylococci, and vancomycin resistant enterococci. This activity is combined with antiinflammatoric activity and low cytotoxicity.

Immunomodulation of RAW264.7 macrophages by GLIS, a proteopolysaccharide from Ganoderma lucidum.

The immunomodulatory effect of Ganoderma lucidum immunomodulating substance (GLIS) on macrophages has been investigated as part of on-going research into the anti-cancer properties of Ganoderma lucidum. Proliferation of bone marrow macrophages (BMMs) was enhanced by GLIS in a dose-dependent manner. Microscopic examination revealed that numerous GLIS-treated RAW264.7 macrophages were enlarged and formed pseudopodia. Exposure of RAW264.7 macrophages to GLIS resulted in significant increases in NO production, induction of cellular respiratory burst activity, and increased levels of IL-1beta, IL-12p35 and IL-12p40 gene expression. Our data indicate that GLIS activates the immune system by modulating cytokine production.

Cytotoxicity of a new toothpaste based on an ion exchange resin mixture.

PURPOSE: To evaluate the effects for cytotoxicity of two dentifrices: a toothpaste commercially available (Crest Extra-Whitening toothpaste) and a new experimental toothpaste based on a mixture of ion-exchange resins (named NMTD) that supplies calcium, fluoride, phosphate and zinc ions. METHODS: Cultures of mouse fibroblasts cells L929 were used in a MTT assay for in vitro cytotoxicity of the dentifrices. Cells were cultured in Eagle's minimal essential medium supplemented with 10% fetal bovine serum. Cultures were incubated at 37 degrees C in a humidified atmosphere of 5% CO2 and collected by tripsinization (0.05% trypsin/0.5mM EDTA). A 96-well microplate method was employed for the MTT colorimetric assay. Positive control consisted of 10 microl of phenol in 5 ml of 6% media, a dose that produces zero percent cell survival. Negative control was prepared by adding 0.5 ml of HBSS to 4.5 ml of 6% media. The plates were incubated for 24 and 48 hours at 37 degrees C in a 5% CO2 atmosphere. RESULTS: Means and standard deviations of absorbance values for each group and percentage inhibitory dosage (%ID) for each test material were calculated. None of the dentifrices resulted in a percentage of inhibition higher than 50% and did not observe marked increases in cytotoxicity with time of incubation. The positive control gave almost zero percent cell survival, whereas the negative control gave a hundred percent cell survival. Analysis of the results indicated that test dentifrice dose had no significant effect towards the cell viability (P<0.05).

Effects of CO2 laser treatment on fibroblast attachment to root surfaces. A scanning electron microscopy analysis.

BACKGROUND: The aim of this study was to analyze the CO2 laser effects on root surfaces affected by periodontal disease in comparison to scaling and root planing for fibroblast attachment. METHODS: Thirty single-rooted human teeth extracted because of advanced periodontal disease were included in this study. A total of 60 specimens, obtained from all selected teeth, were randomly assigned to 3 groups: 1) control (untreated); 2) hand scaling and root planing (SRP); or 3) laser (CO2 defocused pulsed) and ultrasonic scaling. All the specimens were incubated in Petri dishes with fibroblast suspension, and then observed by scanning electron microscopy (SEM). RESULTS: The control group showed the lowest number of attached cells, with no tightly attached fibroblasts. The laser plus scaling group showed the highest number of attached fibroblasts, with the tightly attached fibroblast prevailing. The laser-treated and scaled root specimens did not show any damage or morphologic alteration of the root surfaces. CONCLUSION: CO2 laser treatment in defocused, pulsed mode with a low power of 2W combined with mechanical instrumentation constitutes a useful tool to condition the root surface and increase fibroblast attachment to root surfaces.

A histidine residue in the extracellular N-terminal domain of the GABA(A) receptor alpha5 subunit regulates sensitivity to inhibition by zinc.

The divalent cation zinc is abundant in the brain, particularly in the mossy fibers of the hippocampus. Recent evidence suggests that zinc is packaged into some synaptic vesicles in this region and can be co-released with neurotransmitter. Zinc inhibits the activity of GABA(A) receptors and the sensitivity of the receptor to zinc is influenced by its alpha subunit subtype composition. The alpha4, alpha5 and alpha6 subunits confer greater sensitivity to zinc than receptors containing other alpha subunits. The alpha4 and alpha5 subunits are highly expressed in hippocampal neurons, and likely mediate any effects of zinc on GABAergic neurotransmission in this area. The alpha5 subunit contains a unique histidine residue in the N-terminal extracellular domain while the other alpha subunits have an aspartate residue in this location. Point mutations were created to exchange the histidine and aspartate residues of the alpha1 and alpha5 subunits. Receptors containing the mutated alpha5((H195D)) subunit had reduced sensitivity to zinc, while alpha1((D191H))beta3gamma2L receptors had increased sensitivity to zinc, similar to the alpha5beta3gamma2L wild type receptors. These findings indicate that histidine195 of the alpha5 subunit plays an important role in determining the sensitivity of recombinant GABA(A) receptors to zinc.

Expression of biologically active human tumor necrosis factor-alpha in transgenic potato plant.

We report the successful insertion of the cDNA of human tumor necrosis factor-alpha (HuTNF-alpha) into the genome of potato plant species, Solanum tuberosum, using Agrobacterium tumefacience-mediated transformation. HuTNF-alpha is a known and essential cytokine mediating host defense against tumors and infectious diseases and an immunomodulating agent. To enhance the accumulation of foreign gene product expression in plant cells, the molecular design of the constructed HuTNF-alpha is presented. Transcription and translation of TNF-alpha in transformants were confirmed by Northern blot, RT-PCR, ELISA, and Western blot, respectively. Expression of the bioactive HuTNF-alpha in plant cells was confirmed by way of the cytotoxic effect of the extract obtained from the transformants against murine L929 cells. We think that the expression level of HuTNF-alpha (15 microg/g potato plant tissue) obtained in the present study may be sufficient to induce responses/effects similar to those generated by TNF-alpha in human milk administered orally. We believe that the TNF-alpha expressed in edible potato plants has tremendous potential for clinical use in the areas of medicine and veterinary science. Its usefulness and applicability, therefore, need to be fully explored.

Critical role for activation of antigen-presenting cells in priming of cytotoxic T cell responses after vaccination with virus-like particles.

Virus-like particles (VLPs) are known to induce strong Ab responses in the absence of adjuvants. In addition, VLPs are able to prime CTL responses in vivo. To study the efficiency of this latter process, we fused peptide p33 derived from lymphocytic choriomeningitis virus to the hepatitis B core Ag, which spontaneously assembles into VLPs (p33-VLPs). These p33-VLPs were efficiently processed in vitro and in vivo for MHC class I presentation. Nevertheless, p33-VLPs induced weak CTL responses that failed to mediate effective protection from viral challenge. However, if APCs were activated concomitantly in vivo using either anti-CD40 Abs or CpG oligonucleotides, the CTL responses induced were fully protective against infection with lymphocytic choriomeningitis virus or recombinant vaccinia virus. Moreover, these CTL responses were comparable to responses generally induced by live vaccines, because they could be measured in primary ex vivo (51)Cr release assays. Thus, while VLPs alone are inefficient at inducing CTL responses, they become very powerful vaccines if applied together with substances that activate APCs.

Effects of combined treatment with 40 degrees C hyperthermia and bleomycin on the accumulation of heat shock protein in murine L cells.

Our research group has reported the enhanced cytotoxicity of combined treatment with bleomycin (BLM) and low hyperthermia at 40 degrees C, using murine L cells, and suggested that post-heating could inhibit BLM-induced sublethal damage repair. For further understanding of the involved mechanisms, we subsequently investigated the kinetics of the cellular accumulations of inducible 72-kDa heat shock protein (hsp72) after 40 degrees C hyperthermia and/or BLM treatment using the same cell line. Western blot analysis showed significantly enhanced accumulation of hsp72 after a low hyperthermia at 40 degrees C for 40, 105 or 180 min, and no significant enhancement of it after exposure to 10 microg/ml BLM at 37 degrees C for either 40 or 105 min. When the cells were heated in the presence of BLM, the accumulations of hsp72 were markedly suppressed, with the maxima of hsp72 accumulation decreasing to 38% and 63% of those induced by hyperthermia alone for 40 or 105 min, respectively. On the other hand, sequential treatment with hyperthermia either before or after BLM treatment did not show significant alteration of the heat-induced accumulations of hsp72. It was demonstrated that BLM was necessary during heating to effectively suppress the heat-induced accumulation of hsp72. This study indicates that the suppression of heat-induced accumulation of hsp72 by BLM may partially contribute to enhance cytotoxicity of the simultaneous treatment of 40 degrees C hyperthermia and BLM.