Recombinant interferon α-2a in combination with dacarbazine in the treatment of metastatic malignant melanoma: analysis of long-term responding patients

Thirty-four evaluable patients with metastatic malignant melanoma were entered into a phase-II study designed to assess the response rate and analyze the long-term therapeutic efficacy of recombinant interferon (rIFN) -2a and dacarbazine. Patients received 14 days of daily subcutaneous r-IFN-2a (3×10⁶ IU/day), followed by 9×10⁶ IU on alternate days, as long as objective response lasted, in combination with i.v. dacarbazine started on day 7 (400 mg/m²) and repeated every 21 days (dacarbazine doses were escalated to 800 mg/m²). In 11 patients, 6 complete (17.6%) and 5 partial (14.7%) responses were seen, with an overall response rate of 32.3% (95% confidence interval: 16%–48%). The median survival time of the responding patients was significantly better than that of patients with progressive disease (P=0.01) and the median response time of the patients showing complete response was longer than that of the partially responding patients (14 and 7 months respectively,P=0.06).     

Phase II study of recombinant interferon alpha-C in patients with metastatic renal cell carcinoma

Recombinant interferon alpha-C is a new strain of the alpha interferon family. It was given to 33 patients with measurable metastatic renal cell carcinoma of whom 31 were evaluable. Protocol consisted of 3 million U/d for 2 weeks, then 3 million U/m2 every other day until progression. No complete response was observed. Three patients (9.7%) had partial response for a mean duration of 5.6 months and eight patients (25.8%) were stabilized for a mean of 4.3 months. Responsive sites were mainly lung, bone, and kidney, while side effects were generally mild. better results were observed in previously nephrectomized patients who had not received chemotherapy or hormonotherapy for recurrent or metastatic disease (p less than 0.05), and also in patients with a brief disease-free interval and short delay from presenting symptoms of the primary tumor until interferon treatment (p less than 0.05). Median survival was significantly longer in responders than in progressors (p less than 0.05). We suggest that the efficacy of recombinant interferon alpha-C in a low-dose regime versus other types of interferon as first-line therapy for inoperable, metastatic, or locally recurrent renal cell carcinoma should be investigated in a prospective, controlled, randomized study.     

Distribution of surface charge and concanavalin a-binding sites on normal and malignant transformed cells

Measurement of the rate of agglutination with the positively charged poly- -lysine of normal lymphocytes, Moloney-virus-transformed lymphoma cells, normal fibroblasts and SV40 transformed fibroblasts, has shown that the normal cells were agglutinated at a higher rate than the transformed cells. The labeling density of cationized ferritin in electron micrographs of sectioned cells, also indicated a higher charge density for the normal lymphocytes and fibroblasts. The normal cells showed a more regular clustered distribution of cationized ferritin than the transformed cells, and pre-fixation of cells with glutaraldehyde before labeling with cationized ferritin resulted in a random distribution in both types of cells. The transformed cells had a higher agglutinability than the normal cells by Concanavalin A (ConA) and this difference was also found after treatment of the cells with neuraminidase. Labeling with ConA-ferritin showed the same distribution on the sectioned normal and transformed cells. The results indicate that there was a difference in the redistribution of surface charge by cationized ferritin in normal and transformed cells and that there was no detectable difference in redistribution of ConA-binding sites with ConA-ferritin.     

Rotational relaxation time of concanavalin A bound to the surface membrane of normal and malignant transformed cells

Fluorescence polarization was used to determine the rotational relaxation time of fluorescein conjugated concanavalin A bound to the surface membrane of normal and malignant cells. The cells used were normal lymphocytes and malignant lymphoma cells, as examples of cells that are in suspension in vivo, and normal and simian virus 40-transformed fibroblasts, as examples of cells that form a solid tissue. The relaxation time of F-concanavalin A² in phosphate-buffered saline, pH 7.2, at 24 °C was 58 nseconds. Under the same conditions, the relaxation times of F-concanavalin A bound to cells were: 70 nseconds for normal lymphocytes, 160 nseconds for malignant lymphoma cells, 120 nseconds for normal fibroblasts and 73 nseconds for simian virus 40-transformed fibroblasts. When cells were treated with trypsin or glutaraldehyde before adding F-concanavalin A, trypsin treatment produced a decrease, whereas glutaraldehyde fixation produced an increase of these values. Inhibition of cap formation of concanavalin A binding sites on normal lymphocytes by treating the cells with sodium azide did not change the rotational relaxation time of concanavalin A bound to the cells. These results indicate that the carbohydrate-containing structures on the cells that bind concanavalin A are mobile. In cells that are in suspension in vivo, malignant transformation is associated with reduction in mobility of these sites. However, in cells that form a solid tissue, malignant transformation is associated with an increase in mobility of these sites. Determination of the rotational relaxation time of fluorescent probes bound to specific sites on cell membranes can thus be used to quantitate receptor mobility in relation to cell behaviour.     

Microviscosity parameters and protein mobility in biological membranes.

A fluorescence polarization technique with 1,6-diphenyl 1,3,5-hexatriene as a probe were employed to determine the microviscosity, n, in liposomes and biological membranes of different cholesterol to phospholipid mol ratio. From the temperature profile of n the flow activation energy, deltaE, and the unit flow volume, V, were derived. The increase of cholesterol/phospholipid ratio in liposomes is followed by a marked increase in n and a decrease in both deltaE and V. Liposomes of the same phospholipid composition as human erythrocyte membranes display in the extreme cases of cholesterol/phospholipid ratios 0 and 1.4 the values of n(25 degrees C) = 1.8 and 9.1 P, and deltaE = 15.0 and 6.5 kcal/mol, respectively. For most membranes studied the fluorescence polarization characteristics and the corresponding n values are similar to those obtained with these liposomes when the cholesterol/phospholipid level of the liposomes and the membranes were the same. However, unlike in liposomes deltaE of all membranes is in the narrow range of 6.5-8.5 kcal/mol, regardless of its cholesterol/phospholipid level. It is plausible that this is a general characteristic of biological membranes which originates from the vertical movement of membrane proteins to an equilibrium position which maintains constant deltaE and V values. This type of movement should affect the interrelation between lipid fluidity and protein mobility. Lipid microviscosity and the degree of rotational mobility of concanavalin A receptor sites in cell membranes were therefore determined. The examined cells were normal and malignant fibroblasts, as an example of cells that form solid tumours in vivo, and normal and malignant lymphocytes, as an example of cells that form ascites tumours in vivo. In both cell systems, opposite correlations between the lipid fluidity and the mobility of concanavalin A receptors were observed. In the fibroblasts the malignant cells possess a lower lipid fluidity but a higher receptor mobility, whereas in the lymphocytes the malignant cells possess a higher lipid fluidity but a lower receptor mobility. Thus, in these cell systems the degree of rotational mobility of concanavalin A receptors increases upon decreasing the lipid fluidity and decreases upon increasing the fluidity of the lipid core. This dynamic feature is in line with the above proposal according to which the concanavalin A receptor sites become more exposed to the aqueous surrounding upon increasing the microviscosity of the lipid layer and vice versa.     

Two types of FtsH protease subunits are required for chloroplast biogenesis and Photosystem II repair in Arabidopsis

FtsH protease is important in chloroplast biogenesis and thylakoid maintenance. Although bacteria contain only one essential FTSH gene, multiple genes exist in cyanobacteria and higher plants. However, the functional significance of FTSH multiplication in plants is unclear. We hypothesized that some FTSH genes may be redundant. To test this hypothesis, we generated double mutant combinations among the different FTSH genes in Arabidopsis thaliana. A double mutant of ftsh1 and ftsh8 showed no obvious phenotypic alterations, and disruption of either FTSH1 or FTSH5 enhanced the phenotype of the ftsh2 mutant. Unexpectedly, new phenotypes were recovered from crosses between ftsh2 and ftsh8 and between ftsh5 and ftsh1, including albinism, heterotrophy, disruption of flowering, and severely reduced male fertility. These results suggest that the duplicated genes, FTSH1 and FTSH5 (subunit type A) and FTSH2 and FTSH8 (subunit type B), are redundant. Furthermore, they reveal that the presence of two types of subunits is essential for complex formation, photosystem II repair, and chloroplast biogenesis.