Helicobacter pylori urease suppresses bactericidal activity of peroxynitrite via carbon dioxide production

Helicobacter pylori can produce a persistent infection in the human stomach, where chronic and active inflammation, including the infiltration of phagocytes such as neutrophils and monocytes, is induced. H. pylori may have a defense system against the antimicrobial actions of phagocytes. We studied the defense mechanism of H. pylori against host-derived peroxynitrite (ONOO(-)), a bactericidal metabolite of nitric oxide, focusing on the role of H. pylori urease, which produces CO(2) and NH(3) from urea and is known to be an essential factor for colonization. The viability of H. pylori decreased in a time-dependent manner with continuous exposure to 1 microM ONOO(-), i.e., 0.2% of the initial bacteria remained after a 5-min treatment without urea. The bactericidal action of ONOO(-) against H. pylori was significantly attenuated by the addition of 10 mM urea, the substrate for urease, whereas ONOO(-)-induced killing of a urease-deficient mutant of H. pylori or Campylobacter jejuni, another microaerophilic bacterium lacking urease, was not affected by the addition of urea. Such a protective effect of urea was potentiated by supplementation with exogenous urease, and it was almost completely nullified by 10 microM flurofamide, a specific inhibitor of urease. The bactericidal action of ONOO(-) was also suppressed by the addition of 20 mM NaHCO(3) but not by the addition of 20 mM NH(3). In addition, the nitration of L-tyrosine of H. pylori after treatment with ONOO(-) was significantly reduced by the addition of urea or NaHCO(3), as assessed by high-performance liquid chromatography with electrochemical detection. These results suggest that H. pylori-associated urease functions to produce a potent ONOO(-) scavenger, CO(2)/HCO(3)(-), that defends the bacteria from ONOO(-) cytotoxicity. The protective effect of urease may thus facilitate sustained bacterial colonization in the infected gastric mucosa.     

Comparison of an immunochromatography test with multiplex reverse transcription-PCR for rapid diagnosis of respiratory syncytial virus infections

A new commercial rapid 10-min one-step immunochromatography (IC) test, SAS RSV test, was compared to another IC test, Directigen EZ RSV, employing RT-PCR as the "gold standard" for detecting respiratory syncytial virus. Of 102 clinical samples, 79 were positive by RT-PCR, 66 (82.5%) were positive with the SAS RSV test, and 55 (69.6%) were positive with Directigen EZ RSV. The specificity of the new test was 91.3% (21 of 23), similar to that of Directigen EZ RSV (100% [23 of 23]). This test performs well enough to be used for patient care.     

Direct in vivo protein transduction into a specific restricted brain area in rats

Attempts at protein transduction into specific restricted brain areas have remained unsuccessful. We attempted targeted, direct in vivo protein transduction by microinjecting beta-galactosidase (beta-gal) with hemagglutinating virus of Japan envelope (HVJ-E) vector into the rat nucleus tractus solitarius (NTS). The medulla oblongata including the NTS was removed 6h post-injection and cryostat sections were histochemically stained to detect beta-gal enzymatic activity. beta-gal-positive cells were present in these sections as was beta-gal activity determined by colorimetric analysis. beta-gal-positive cells were not present in the rats microinjected only beta-gal protein without HVJ-E vector. Our findings suggest that direct in vivo protein transduction into specific restricted brain areas is possible. The type of targeted delivery system we present may have wide applications in the administration of therapeutic proteins to the central nervous system.     

Graphic and movie illustrations of human prenatal development and their application to embryological education based on the human embryo specimens in the Kyoto collection.

Morphogenesis in the developing embryo takes place in three dimensions, and in addition, the dimension of time is another important factor in development. Therefore, the presentation of sequential morphological changes occurring in the embryo (4D visualization) is essential for understanding the complex morphogenetic events and the underlying mechanisms. Until recently, 3D visualization of embryonic structures was possible only by reconstruction from serial histological sections, which was tedious and time-consuming. During the past two decades, 3D imaging techniques have made significant advances thanks to the progress in imaging and computer technologies, computer graphics, and other related techniques. Such novel tools have enabled precise visualization of the 3D topology of embryonic structures and to demonstrate spatiotemporal 4D sequences of organogenesis. Here, we describe a project in which staged human embryos are imaged by the magnetic resonance (MR) microscope, and 3D images of embryos and their organs at each developmental stage were reconstructed based on the MR data, with the aid of computer graphics techniques. On the basis of the 3D models of staged human embryos, we constructed a data set of 3D images of human embryos and made movies to illustrate the sequential process of human morphogenesis. Furthermore, a computer-based self-learning program of human embryology is being developed for educational purposes, using the photographs, histological sections, MR images, and 3D models of staged human embryos.     

Clinical study on the serum carcinoembryonic antigen, immunosuppressive acidic protein and C-reactive protein levels in patients with adult T cell leukemia

Serum carcinoembryonic antigen (CEA), immunosuppressive acidic protein (IAP) and C-reactive protein (CRP) levels were measured in patients with adult T cell leukemia (ATL) in order to clarify their significance in this disease. Mean (+/- SD) serum CEA levels in 11 patients with acute ATL (3.1 +/- 1.3 ng/ml) and 7 patients with smoldering ATL (3.1 +/- 0.5 ng/ml) were significantly higher than in sera of 222 healthy controls (2.4 +/- 1.3 ng/ml). However, the levels in 7 patients with chronic ATL and healthy controls showed no differences. On the other hand, mean (+/- SD) serum IAP levels in patients with acute ATL (928 +/- 395 micrograms/ml), chronic ATL (487 +/- 125 micrograms/ml) and smoldering ATL (429 +/- 90 micrograms/ml) were significantly higher than in sera of healthy controls (359 +/- 103 micrograms/ml). However, the levels in patients with smoldering ATL and healthy controls showed no differences. Serum IAP levels in crisis in chronic and smoldering ATL were similar to those in patients with acute ATL. 85% of ATL patients with IAP levels above 500 micrograms/ml had CRP levels above 1+. Serum CEA, IAP and CRP levels were serially measured in a number of patients. Serum IAP and CRP levels reflected each patient's clinical course more than serum CEA levels. Overall the simultaneous measurements of serum CEA, IAP and CRP levels revealed a potential usefulness for determination of ATL subtype, and serum IAP and CRP levels may provide a way to evaluate the effectiveness of chemotherapy.     

Dedifferentiated adenoid cystic carcinoma: a clinicopathologic study of 6 cases.

Dedifferentiated adenoid cystic carcinomas are a recently defined, rare variant of adenoid cystic carcinomas characterized histologically by two components: conventional low-grade adenoid cystic carcinoma and high-grade "dedifferentiated" carcinoma. We examined six cases and analyzed their clinicopathologic profiles, including immunohistochemical features and p53 gene alterations. The 6 patients (3 men and 3 women) had a mean age of 46.8 years (range, 34-70 y). The mean size of the tumors was 3.5 cm (range, 1.7-6 cm). The submandibular gland, maxillary sinus, and nasal cavity were involved in 2 cases each. Postoperatively, 5 patients had local recurrence and 5 developed metastatic disease. Five patients died of disease at a mean of 33.7 months after diagnosis (range, 6-69 mo), and one other was alive with disease at 60 months. Histologically, the conventional low-grade adenoid cystic carcinoma component of the tumors consisted of a mixture of cribriform and tubular patterns with scant solid areas. The high-grade dedifferentiated carcinoma component was either a poorly differentiated adenocarcinoma (4 cases) or undifferentiated carcinoma (2 cases). Three tumors were studied immunohistochemically. Myoepithelial markers were expressed in low-grade adenoid cystic carcinoma but not in the dedifferentiated component. In 2 cases, diffusely positive p53 immunoreactivity together with HER-2/neu overexpression was restricted to the dedifferentiated component. Loss of pRb expression was demonstrated only in the dedifferentiated component of the 1 other case. The Ki-67-labeling index was higher in the dedifferentiated component than in the low-grade adenoid cystic carcinoma component. Furthermore, molecular analysis of 2 cases demonstrated the loss of heterozygosity at p53 microsatellite loci, accompanied by p53 gene point mutation, only in the dedifferentiated carcinoma component of 1 case, which was positive for p53 immunostaining. These results indicate that dedifferentiated adenoid cystic carcinoma is a highly aggressive tumor. Because of frequent recurrence and metastasis, the clinical course is short, similar to that of adenoid cystic carcinomas with a predominant solid growth pattern. Limited evidence suggests that p53 abnormalities in combination with HER-2/neu overexpression or loss of pRb expression may have a role in dedifferentiation of adenoid cystic carcinoma.     

Rapid screening of point mutations of the Neisseria gonorrhoeae parC gene associated with resistance to quinolones.

To detect quinolone resistance-associated mutations within the Asp-86, Ser-87, Ser-88, and Glu-91 codons of the Neisseria gonorrhoeae parC gene, we developed a rapid and simple assay based on amplification of the regions of the parC gene containing the mutations sites by PCR and digestion of the PCR products with restriction enzymes. By using the method of primer-specified restriction site modification, artificial SalI, PstI, EcoRI, and HinfI restriction sites were created in the regions containing the Asp-86, Ser-87, Ser-88, and Glu-91 codons, respectively. The mutations generating alterations at Asp-86, Ser-87, Ser-88, and Glu-91 were detected as failures of SalI, PstI, EcoRI, and HinfI to digest the respective PCR products. Fifty-five clinical strains of N. gonorrhoeae were examined for mutations in the parC gene by this assay. Appropriate mutations at either the Asp-86, Ser-87, Ser-88, or Glu-91 codon were detected in each of 11 strains in which a mutation had previously been observed by DNA sequencing. This rapid and simple assay could be a useful device for screening genetic alterations in the parC gene associated with resistance to quinolones in N. gonorrhoeae.     

Protein kinase C (PKC) activity and PKC messenger RNAs in human pituitary adenomas.

Protein kinase C (PKC) is involved in the differentiation and growth regulation of a variety of tissues including anterior pituitary gland cells. To determine the distribution of PKC in different types of adenomas, PKC activity was analyzed in human pituitary tumors and the effects of hypothalamic hormone stimulation on PKC activity were examined in cultured adenoma cells. Gonadotroph (LH/FSH) and null cell adenomas had significantly higher levels of particulate, soluble, and total PKC activity compared with growth hormone (GH) adenomas (P < 0.05). Chronic stimulation of null cell adenomas with gonadotropin hormone-releasing hormone or of one GH adenoma with GH-releasing hormone for 7 days did not significantly alter total PKC activity in pituitary cells cultured in serum-free medium. Localization of the calcium-dependent PKC isozymes (alpha, beta and gamma) by immunohistochemistry and in situ hybridization revealed predominantly PKC alpha in all adenomas and variable expression of PKC beta and gamma in some tumors. When the calcium-independent PKC isozymes (delta, epsilon, and zeta) were localized by in situ hybridization, normal and neoplastic pituitaries expressed abundant mRNA for PKC epsilon, whereas some tumors and one normal pituitary had a few cells positive for PKC zeta mRNA as evaluated by grain density and the number of cells labeled. These results indicate that there is a variable distribution of PKC mRNA isozymes in human pituitary adenomas and that normal pituitaries and pituitary adenoma cells express the mRNA for both the calcium-dependent and some of the calcium-independent PKC isozymes. Chronic treatment with the hypothalamic gonadotropin hormone-releasing hormone and GH-releasing hormone, which increased LH/FSH and GH secretion, respectively, did not increase PKC activity in cultured adenoma cells. The presence of calcium-dependent and calcium-independent PKC isozymes in normal and neoplastic pituitary cells indicates that PKC probably plays a major role in signal transduction in the human pituitary adenomas examined in this study.     

A case of sarcomatoid carcinoma of the thymus

A 57-year-old woman presented with a 10×10 cm anterior mediations mass. The tumor had Invaded the pericardium, both lungs and the left brachiocephallc vein, and was treated by partial resection and postoperative radiation therapy. Pathological examination of the tumor revealed squamous cell carcinoma with a spindle cell sarcomatous component. Immunohistochemically, keratin and epithelial membrane antlgen were posltive In both the spindle cell sarcomatous areas and the squamous cell carcinomatous area and thus, a diagnosis of thymic carcinoma of sarcomatoid type was made. The patient died of recurrent disease 1 year after surgery. This case is the seventh reported In the English literature Because of the poor outcome, adjuvant therapy is recommended.     

Direct evidence for clonal destruction of allo-reactive T cells in the mice treated with cyclophosphamide after allo-priming.

It has previously been reported that a single i.p. injection of 200 mg/kg cyclophosphamide (CP) 2 days after priming with 10(8) donor spleen cells (SC) leads to donor-specific skin allograft tolerance in H-2 compatible, multiminor antigen incompatible murine strain combinations. It is speculated that the i.v. injection of donor cells may result in synchronized proliferation of donor-reactive host T cells and subsequently administered CP may specifically destroy these proliferating T cells in the periphery. Although this unique action of CP is considered to be a principal mechanism in this method, direct evidence has not yet been obtained. In the present article, this in vivo destructive effect of CP is clearly demonstrated by assessing detailed kinetics of host-derived blastoid T cells and donor (Mls-1a)-reactive V beta 6+ T cells in the model system of C3H mice rendered tolerant to AKR. Frequencies of the blastoid cells and V beta 6+ cells, which increased as a result of AKR priming, decreased rapidly with the administration of CP. C3H mice, which received AKR SC alone, also exhibited partial deletion of V beta 6+ T cells, but both tempo and magnitude of decrease in the frequency of V beta 6+ cells were quite different from those of the C3H mice given AKR SC and CP, which showed more rapid and profound elimination of V beta 6+ T cells. In accordance with these kinetic studies, in vitro proliferative response to Mls-1a antigens was greatly impaired in mice treated with SC and CP, whereas a low but appreciable response was detected in mice given SC alone.(ABSTRACT TRUNCATED AT 250 WORDS)