Activation of murine T cells by staphylococcal enterotoxin E: requirement of MHC class II molecules expressed on accessory cells and identification of V beta sequence of T cell receptors in T cells reactive to the toxin

We investigated a mechanism leading to activation of murine T cells by staphylococcal enterotoxin E (SEE). L cells transfected with I-Ab genes but not control L cells supported IL-2 production by SEE-induced C57BL/6 T lymphoblasts upon restimulation with SEE. mAb to I-Ab markedly inhibited the above response. Flow cytometric analyses showed that SEE-induced C57BL/6 T lymphoblasts are composed of both CD4+ T cells and CD8+ T cells, and that larger parts of them bore V beta 11 (40-75%). mAb to V beta 11 markedly inhibited the SEE-induced proliferative response and IL-2 production by T cells. Analysis of SEE-induced IL-2 production in spleen cells from various mouse strains showed that C57BL/6 and B10.A(4R) mice (I-E, not expressed; V beta 11+ T cells, normally generated) are highly responsive to SEE. In contrast, BALB/c, C3H/HeN, (C57BL/6 x BALB/c or C3H/HeN) F1 mice (I-E, normally expressed and V beta 11+ T cells, deleted), and SJL and C57L mice (V beta 11 genes, deleted) are weakly responsive to SEE. The results indicate that SEE activates mainly T cells bearing V beta 11 in physical association with MHC class II molecules expressed on AC. In addition, the results indicate that SEE activates both CD4+ T cells and CD8+ T cells.     

Koningic Acid (a Potent Glyceraldehyde-3-Phosphate Dehydrogenase Inhibitor)-Induced Fragmentation and Condensation of DNA in NG108-15 Cells

Abstract: We examined nitric oxide (NO)-induced cell death in NG108-15 cells using NO donors. Both sodium nitroprusside (SNP) and S -nitroso- N -acetylpenicillamine caused lactate dehydrogenase (LDH) leakage from NG108-15 cells. NO is known to increase the amount of radioisotopic labeled glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the presence of [³²P]NAD and to inhibit the enzyme activity. To clarify the relationship between the NO-induced inhibition of GAPDH activity and cell death, we studied the effect of koningic acid (KA), a potent selective inhibitor of GAPDH. Both SNP and KA elicited LDH leakage, chromosomal condensation, and fragmentation of nuclei in NG108-15 cells. Gel electrophoretic analysis of cellular DNA extracted from SNP- and KA-treated cells revealed the internucleosomal DNA fragmentation typical of apoptosis in these cultures. The results suggested that in NG108-15 cells, (a) the inhibition of GAPDH activity results in apoptosis and (b) SNP-induced cell death is partly due to the NO-induced inhibition of GAPDH, perhaps by stimulating the binding of NAD to GAPDH.     

Smoking-reIated DNA adducts and genetic polymorphism for metabolic enzymes in human lymphocytes

Smoking-related aromatic DNA adducts in lymphocytes were measured from smokers (n = 76), ex-smokers (n = 25) and non-smokers (n = 56) by the ³²P-postlabelling method, to clarify whether a genetic polymorphism for metabolic enzymes could explain the inter-individual variation of DNA adduct levels. Adduct levels were compared with respect to smoking status and polymorphic genotypes of cytochrome P4501A1 (CYP1A1) and glutathione S-transferase M1 (GTSM1). The mean adduct level (1.24 per 10⁸ nucleotides) in smokers was significantly higher than that (0.85 per 10⁸) in non-smokers. Although we expected higher adduct levels in the CYP1A1 variant or GSTM1 null subjects, the adduct level in 'GSN1 nulls' was significantly lower than that in 'GSTM1 presents' among smokers. DNA adduct levels had significant positive correlations with smoking indices such as number of cigarettes or smoking years in all subjects. In smokers only, however, no correlation was found, because there were negative correlations between adduct levels and smoking dose in GSTM1 null genotypes. CYP1A1 genotypes had no effects on adduct levels.     

Detection of Ubiquitinated Dermcidin in Gingival Crevicular Fluid in Periodontal Disease

Periodontal disease is an inflammatory disease caused by gram-negative bacteria, such as Porphyromonas gingivalis and Actinobacllus actinomycetemcomitans. Antimicrobial peptides kill organisms, such as gram-negative and gram-positive bacteria, mycobacteria, enveloped viruses, and fungi. We previously identified the antimicrobial peptide dermcidin (DCD) in the gingival crevicular fluid (GCF) using proteomic analyses. Moreover, western blot analysis revealed that the molecular weights of GCF protein bands considerably varied (approximately 27 kDa). We attempted to explore the considerable variation in the molecular weights of protein bands using on-membrane digestion and liquid chromatography–tandem mass spectrometry (LC–MS/MS) analyses. We examined ubiquitin among the DCD-interacting proteins. In immunoprecipitation experiments, ubiquitinated DCD was detected by western blotting and by immunoprecipitation with an antibody against DCD and mono- or poly-ubiquitinated proteins. These analyses indicated the possible involvement of the ubiquitination reaction. Ubiquitinated DCD may protect against periodontal bacterial pathogen invasion in GCF.     

Reducing Short-Wavelength Blue Light in Dry Eye Patients with Unstable Tear Film Improves Performance on Tests of Visual Acuity

PurposeTo investigate whether suppression of blue light can improve visual function in patients with short tear break up time (BUT) dry eye (DE).MethodsTwenty-two patients with short BUT DE (10 men, 12 women; mean age, 32.4 ± 6.4 years; age range, 23–43 years) and 18 healthy controls (10 men, 8 women; mean age, 30.1 ± 7.4 years; age range, 20–49 years) underwent functional visual acuity (VA) examinations with and without wearing eyeglasses with 50% blue light blocked lenses. The functional VA parameters were starting VA, functional VA, and visual maintenance ratio.ResultsThe baseline mean values (logarithm of the minimum angle of resolution, logMAR) of functional VA and the visual maintenance ratio were significantly worse in the DE patients than in the controls (P < 0.05), while no significant difference was observed in the baseline starting VA (P > 0.05). The DE patients had significant improvement in mean functional VA and visual maintenance ratio while wearing the glasses (P < 0.05), while there were no significant changes with and without the glasses in the control group (P > 0.05),ConclusionsProtecting the eyes from short-wavelength blue light may help to ameliorate visual impairment associated with tear instability in patients with DE. This finding represents a new concept, which is that the blue light exposure might be harmful to visual function in patients with short BUT DE.     

Phenotype of a Temperature-Sensitive, Respiration-Deficient (cyt) Mutant of Yeast

A temperature-sensitive respiration-deficient mutant of yeast lacks hemoproteins and accumulates coproporphyrin III when cultivated at elevated temperatures. Cells grown at 20 C respired normally and contained cytochromes a, b, and c. Cells grown at 35 C showed respiration-deficient mutant characters; they did not respire, lacked cytochromes, and accumulated coproporphyrin III. Addition of protoporphyrin IX or protohemin IX to the culture medium restored the respiratory activity of this mutant during growth at 35 C. The activities of various enzymes, including succinate-2,6-dichlorophenol indophenol (DCPIP), reduced nicotinamide adenine dinucleotide (NADH(2))-DCPIP, succinate-cytochrome c, and NADH(2)-cytochrome c oxidoreductase, and cytochrome oxidase, and the cytochrome c content of cells cultured in various conditions were determined. Changes in the number and structure of mitochondria were associated with changes in respiratory activity.     

Sequence Analysis of the actA Gene of Listeria monocytogenes Isolated from Human

The region encoding proline-rich units of actA genes was amplified from 24 strains of Listeria monocytogenes using polymerase chain reaction (PCR). PCR products of 13 strains showed the expected size of 623 bp, whereas those of 11 strains showed a short size of 518 bp. The shortening of these PCR products resulted from the deletion of one proline-rich unit. These results indicate that ActA proteins are divided into at least two different types which are unrelated to bacterial serotypes.     

Human Nanog pseudogene8 promotes the proliferation of gastrointestinal cancer cells

There is emerging evidence that human solid tumor cells originate from cancer stem cells (CSCs). In cancer cell lines, tumor-initiating CSCs are mainly found in the side population (SP) that has the capacity to extrude dyes such as Hoechst 33342. We found that Nanog is expressed specifically in SP cells of human gastrointestinal (GI) cancer cells. Nucleotide sequencing revealed that NanogP8 but not Nanog was expressed in GI cancer cells. Transfection of NanogP8 into GI cancer cell lines promoted cell proliferation, while its inhibition by anti-Nanog siRNA suppressed the proliferation. Immunohistochemical staining of primary GI cancer tissues revealed NanogP8 protein to be strongly expressed in 3 out of 60 cases. In these cases, NanogP8 was found especially in an infiltrative part of the tumor, in proliferating cells with Ki67 expression. These data suggest that NanogP8 is involved in GI cancer development in a fraction of patients, in whom it presumably acts by supporting CSC proliferation.     

Design, synthesis, and structure-activity relationships of a series of 2-Ar-8-methyl-5-alkylaminoquinolines as novel CRF(1) receptor antagonists

We designed and synthesized a series of 2-Ar-8-methyl-5-alkylaminolquinolines as potent corticotropin-releasing factor 1 (CRF(1)) receptor antagonists. The structure-activity relationships of substituents at each position (R(3), R(5), R(5'), and R(8)) was investigated. By derivatization, three compounds (6, 14b, and 14c) were identified as orally active CRF(1) receptor antagonists.     

Dopamine release via the vacuolar ATPase V0 sector c-subunit,confirmed in N18 neuroblastoma cells,results in behavioral recovery in hemiparkinsonian mice

A 16-kDa proteolipid, mediatophore, in Torpedo electric organs mediates Ca²⁺-dependent acetylcholine release. Mediatophore is identical to the pore-forming stalk c-subunit of the V0 sector of vacuolar proton ATPase (ATP6V0C). The function of ATP6V0C in the mammalian central nervous system is not clear. Here, we report transfection of adeno-associated viral vectors harboring rat ATP6V0C into the mouse substantia nigra, in which high potassium stimulation increased overflow of endogenous dopamine (DA) measured in the striatum by in vivo microdialysis. Next, in the striatum of 6-hydroxydopamine-lesioned mice, a model of Parkinson’s disease (PD), human tyrosine hydroxylase, aromatic l-amino-acid decarboxylase and guanosine triphosphate cyclohydrolase 1, together with or without ATP6V0C, were expressed in the caudoputamen for rescue. Motor performance on the accelerating rotarod test and amphetamine-induced ipsilateral rotation were improved in the rescued mice coexpressing ATP6V0C. [³H]DA, taken up into cultured N18 neuronal tumor cells transformed to express ATP6V0C, was released by potassium stimulation. These results indicated that ATP6V0C mediates DA release from nerve terminals in the striatum of DA neurons of normal mice and from gene-transferred striatal cells of parkinsonian mice. The results suggested that ATP6V0C may be useful as a rescue molecule in addition to DA-synthetic enzymes in the gene therapy of PD.