Proteins of herpesvirus type 2: I. Virion, nonvirion, and antigenic polypeptides in infected cells.

Purified herpesvirus type 2 (HSV-2 (G)) virions were prepared by two cycles of centrifugation on dextran T-10 gradients from cytoplasmic extracts of infected human epidermoid carcinoma (HEp-2) cells. The purity of the preparation was assessed by electron microscopy and by an analysis of the ratio of infectivity to trichloroacetic acid-precipitable counts. The extent of purification by the latter criterion was at least 240-fold with respect to host proteins. Sodium dodecyl sulfate-acrylamide-gel electrophoresis of the proteins from these virion preparations revealed the existence of a minimum of 24 proteins. Forty-seven viral proteins were identified in HSV-2 (G) infected HEp-2 cells by using one or more of the following criteria: (i) an increased rate of synthesis following infection; (ii) precipitation by antisera specific for viral antigens; and (iii) variations in their electrophoretic mobilities or rates of synthesis following infection with different strains of HSV-2. Twenty-four of these viral proteins have electrophoretic mobilities similar to those of the proteins prepared from purified virions. The remaining 23 viral proteins were classified as nonstructural proteins. The 47 viral proteins could be subclassified into four groups on the basis of their kinetics of synthesis, indicating regulation of the amounts of viral protein synthesized. The data are discussed in terms of their relevance to the molecular aspects of productive HSV-2 infection and to the identification of the viral antigens responsible for stimulating the production of neutralizing and complement-fixing antibodies.     

Identification of Rhodococcus fascians cytokinins and their modus operandi to reshape the plant

Decades ago, the importance of cytokinins (CKs) during Rhodococcus fascians pathology had been acknowledged, and an isopentenyltransferase gene had been characterized in the fas operon of the linear virulence plasmid, but hitherto, no specific CK(s) could be associated with virulence. We show that the CK receptors AHK3 and AHK4 of Arabidopsis thaliana are essential for symptom development, and that the CK perception machinery is induced upon infection, underlining its central role in the symptomatology. Three classical CKs [isopentenyladenine, trans-zeatin, and cis-zeatin (cZ)] and their 2-methylthio (2MeS)-derivatives were identified by CK profiling of both the pathogenic R. fascians strain D188 and its nonpathogenic derivative D188–5. However, the much higher CK levels in strain D188 suggest that the linear plasmid is responsible for the virulence-associated production. All R. fascians CKs were recognized by AHK3 and AHK4, and, although they individually provoked typical CK responses in several bioassays, the mixture of bacterial CKs exhibited clear synergistic effects. The cis- and 2MeS-derivatives were poor substrates of the apoplastic CK oxidase/dehydrogenase enzymes and the latter were not cytotoxic at high concentrations. Consequently, the accumulating 2MeScZ (and cZ) in infected Arabidopsis tissue contribute to the continuous stimulation of tissue proliferation. Based on these results, we postulate that the R. fascians pathology is based on the local and persistent secretion of an array of CKs.     

Peritoneal carcinomatosis of unknown primary site in women. A distinctive subset of adenocarcinoma

STUDY OBJECTIVE: To define the clinical features and results of systemic treatment in women with adenocarcinoma of unknown primary site involving predominantly the peritoneal surfaces. DESIGN: Retrospective analysis of 18 patients treated at a single institution between 1978 and 1984. PATIENTS: All 18 women had abdominal carcinomatosis and had no primary site identified at laparotomy. Nine patients had limited residual tumor (maximal tumor diameter, 3 cm or less) after initial cytoreductive surgery, and 9 patients had extensive residual disease. INTERVENTIONS: In general, patients were treated according to standard guidelines for treatment of advanced ovarian carcinoma. All patients had initial laparotomy with attempted cytoreduction; of these 18 patients, 16 subsequently received cisplatin-based chemotherapy. Patients were restaged either clinically (10 patients) or with second-look surgery (8 patients). RESULTS: The median survival for all patients was 23 months. Five patients had complete response to chemotherapy, and three patients remain disease-free 41, 59, and 77 months after diagnosis. Patients with limited residual disease had longer median survival than did those with extensive residual disease (31 months compared with 11 months). CONCLUSIONS: Women with adenocarcinoma of unknown primary site involving predominantly the peritoneal surface should be distinguished from other patients with adenocarcinoma of unknown primary site because they have a more indolent disease course, a higher response rate to systemic therapy, and a chance for long-term, disease-free survival after therapy. Although optimal treatment is undefined, we recommend that these patients be treated using the guidelines established for therapy of advanced ovarian carcinoma, including initial surgical cytoreduction followed by cisplatin-based combination chemotherapy.     

In vivo selection of human tumor cells resistant to monoclonal antibody-Vinca alkaloid immunoconjugates

UCLA-P3 human lung adenocarcinoma cells were grown in nude mice and given repetitive treatments of a monoclonal antibody-Vinca alkaloid immunoconjugate. Although this therapy resulted in a greater than 4-fold reduction in mean tumor mass of the established tumors, some animals experienced a reinitiation of tumor growth after cessation of conjugate treatment. Two such animals were treated again with high doses of monoclonal antibody-Vinca but one of the tumors was no longer regressed by the drug conjugate. The tumor was excised, enzymatically dissociated, and grown in tissue culture. Cultured cells were reimplanted in nude mice and subjected to further therapy with a monoclonal antibody-Vinca conjugate. The resulting tumors were also refractory to the immunoconjugate therapy. This cycle was repeated for a total of three times and resulted in the serial in vivo selection of three conjugate resistant variants. The mechanism responsible for the in vivo resistance of human tumor cells to the monoclonal antibody-Vinca immunoconjugate is unknown but does not appear to involve antigen modulation, altered tumor cell growth rate, or an apparent decrease in tumor targeting in vivo. The resistance was also not accompanied by any detectable elevation in multidrug resistance 1 mRNA or P-glycoprotein expression. Significantly, the resistance pattern was observed only in vivo and was not maintained by cells grown in vitro.     

In vitro photodynamic therapy on melanoma cell lines with phthalocyanine.

Photodynamic therapy (PDT) is a new treatment modality of tumours. The photochemical interactions of sensitizer, light, and molecular oxygen produce singlet oxygen and other forms of active oxygen, such as peroxide, hydroxyl radical and superoxid anion. Phthalocyanine ClAlPcS(2), belonging among the promising second generation of sensitizers, was tested as an inducer of photodamage. We report the production of reactive oxygen species (ROS) and the phototoxicity of ClAlPcS(2) assessed using G361 melanoma cells. A semiconductor laser (lambda=675nm, output power 21mW) was used as a source for evocation of the photodynamic effect. ROS generation and H(2)O(2) release after PDT on G361 cells were detected using probe CM-H(2)DCFDA and recorded by luminescence spectrometer. Viability studies show, that the optimum phototoxic effect tested on G361 melanoma cells was determined in the combination of laser dose of 25Jcm(-2) and phthalocyanine ClAlPcS(2) concentration of 5mug/ml. This combination of phthalocyanine concentration and corresponding radiation dose was lethal for melanoma cells.