Suramin and heparin: aspecific inhibitors of mesoderm induction in the Xenopus laevis embryo.

Xenopus embryos in solutions containing suramin show a dose-dependent decrease in the formation of dorsoanterior structures. Continuous treatment with 1 mM suramin produces embryos without mesodermal derivatives but with mesenchymal cells. Brief immersions of 20 min were used to determine the most sensitive stages and to establish dose-effect curves: a 20 min treatment with 3 mM suramin at stages 7-8.5 produces blastula-like embryos, never classified before, with atypical epidermis, cells full of yolk and mesenchyme in between. The lack of dorsal mesoderm was confirmed by an RNase protection assay with alpha-cardiac actin probe. Heparin also causes a reduction in dorsal structures, but its action is weaker and and there are also strong toxic effects such as superficial cell dissociation. The effect of heparin is dose-dependent and brief immersions show a very sensitive period around stage 6.5. The lowest DAI obtained is 1.5, an extremely microcephalic embryo with forked tail codes, a stocky notochord, and abnormally shaped, abundant neural tissue. Immunofluorescence shows that the distribution of fibronectin-containing fibrils is normal in heparin-treated embryos, whereas there are no such fibrils in suramin-treated embryos at control stage 12.

Morphologic analysis of serous effusion cells by scanning electron microscopy.

The authors used scanning electron microscopy (SEM) to examine the cells obtained from serous effusions of patients affected with a neoplasm or benign disease. Cellular morphology was studied in non-neoplastic effusions to distinguish benign cells from cancer cells with which they are almost always suspended in malignant effusions. Tumor cells differ in some features such as shape, size, arrangement and especially in surface structures (microvilli), the latter being very noticeable when using this method. The investigation enabled us to note several changes in the cell surface that could reflect particular activities of the cells themselves. For this reason we believe that the technique may be considered very useful to gain knowledge of cellular morphology. However, it cannot be considered suitable for making routine diagnostic conclusions.

Immunoendocrine therapy with interleukin-2 (IL-2) and medroxyprogesterone acetate (MPA): a randomized study with or without MPA in metastatic renal cancer patients during IL-2 maintenance treatment after response or stable disease to IL-2 subcutaneous therapy.

AIMS AND BACKGROUND: It is known that interleukin-2 (IL-2) activated cytotoxic lymphocytes require a cell-cell contact to exert their anticancer action. Therefore, the pronounced fibrosis that generally characterizes the neoplastic mass could counteract the action of cytotoxic lymphocytes. Some preliminary studies have shown that progesterone and its analogs may inhibit fibroblast proliferation. On the basis of such evidence, we have designed a clinical study with or without the progestational agent medroxyprogesterone acetate (MPA) in metastatic renal cancer patients in maintenance therapy with IL-2 following response or stable disease (SD) after two cycles of IL-2 subcutaneous immunotherapy, in an attempt to evaluate the influence of MPA on free-from progression (FPP) period. METHODS: The study included 30 consecutive patients who were randomized to receive IL-2 alone (3 million IU twice/day for 5 days/month subcutaneously) or IL-2 plus low-dose MPA (500 mg orally one day/week) without interruption until disease progression. RESULTS: A FPP period longer than 1 year was obtained in 8/14 patients treated with IL-2 plus MPA and in only 3/16 patients treated with IL-2 alone. The difference was statistically significant. On the contrary, no significant difference was seen in the mean number of lymphocytes and eosinophils, which was evaluated monthly. Finally, no hyperglycemic or thromboembolic complications occurred in patients concomitantly treated with MPA. CONCLUSIONS: This preliminary study would suggest that the concomitant administration of low-dose MPA may prolong the FFP period in metastatic renal cancer patients under maintenance therapy with IL-2. A longer follow-up will be required to evaluate the influence of MPA on overall survival.

A randomized study of low-dose interleukin-2 subcutaneous immunotherapy versus interleukin-2 plus interferon-alpha as first line therapy for metastatic renal cell carcinoma.

AIMS AND BACKGROUND: IL-2 given subcutaneously in combination with interferon-alpha 2b (IFN) appears to induce a response rate comparable to that obtained with IL-2 intravenous injection in patients with metastatic renal cell carcinoma (RCC) but with lower toxicity. The role of IFN when combined with IL-2 has however still to be defined. The present study was performed to draw some preliminary conclusions about the effect of IFN in combination with IL-2 in metastatic RCC. METHODS: The study included 30 consecutive patients with metastatic RCC who were randomized to treatment with IL-2 subcutaneous therapy (3 million IU twice/daily for 5 days/week for 6 weeks) or with IL-2 plus IFN (5 million U/m2 subcutaneously thrice weekly). In patients without progressive disease, a second cycle was repeated after a 28-day rest period. RESULTS: No significant difference in partial response rate was found between patients treated with IL-2 alone and those given IL-2 plus IFN (5/15 vs 4/15). Similarly, no difference was seen in the percentage of stable disease (7/15 vs 7/15). Toxicity was higher in patients who received IL-2 plus IFN. Lymphocyte and eosinophil mean increase was higher in patients treated with IL-2 alone than in those treated with IL-2 plus IFN, without however any significant difference. CONCLUSIONS: The present results, which require confirmation in a larger series, indicate that combination with IFN does not increase the efficacy of IL-2 subcutaneous immunotherapy in metastatic RCC but only the toxicity of treatment.

[Experience with rIL2 in the treatment of metastatic renal carcinoma]. / Esperienze con rIL2 nel trattamento del carcinoma renale metastatizzato.

Eight patients with metastatic renal cell carcinoma, all nephrectomized, were treated with rIL2 continuous i.v. infusion or subcutaneous administering (+/- INF). Six patients alive. Continuous infusion is possible event out of Intensive Care Unity. Adverse effects, sometimes serious, rapidly decrease with suspension. Subcutaneous infusion is more easy and with lower adverse effects. We don't know methodic of which can give more security.

[Interferon and vinblastine in advanced renal carcinoma]. / Interferone e vinblastina nel carcinoma renale avanzato.

19 patients with renal adenocarcinoma and mets were treated with INF alfa 2b (10 x 10(6) U/m square s.c. three times weekly) plus VBL (0.1 mg/kg every 3 weeks). We followed up the patients for 12 weeks with monitoring of major parameters. The outcomes have been positive in 16.6% of the cases with mean survival of 20.7 months. We point out the efficacy in lung mets. The outcomes of therapy with INF alfa 2b plus VBL are unsuccessful and we state that an alternative solution could be interleukin 2.

[Immunotherapy with low-dose subcutaneous interleukin-2 in metastatic renal carcinoma]. / Immunoterapia con basse dosi di interleuchina-2 per via sottocutanea nel carcinoma renale metastatico.

The intravenous injection of interleukin-2 (IL-2) has appeared to induce tumor regression in metastatic renal cell carcinoma (RCC). IL-2 given subcutaneously has also appeared to be effective when it is administered in association with interferon-alpha (INF), but with a lower toxicity in comparison to the intravenous route of administration. The present study was carried out to evaluate the efficacy of a subcutaneous immunotherapy with IL-2 alone in metastatic RCC. The study included 30 consecutive patients affected by metastatic RCC, 14 of whom had been pretreated with INF plus vinblastine, while the other 16 patients received IL-2 as a first line therapy of their metastatic disease. IL-2 was given subcutaneously at a dose of 3 million IU twice/day for 5 days/week for 6 consecutive weeks, corresponding to one cycle of immunotherapy. No complete response was obtained. A partial response (PR) was achieved in 10/30 (33%) patients (median duration: 7 months, range 5-25), without any significant difference between patients pretreated with IFN and nonpretreated patients (4/14 vs 6/16). Response rate was significantly higher in nephrectomized patients than in those who did not undergo nephrectomy (10/25 vs 0/5; P < 0.01). Moreover, response rate was significantly higher in patients with performance status (PS) greater than 40% than in those with PS lower than 40% (10/23 vs 0/7; P < 0.01). A stable disease (SD) was obtained in 12/30 (40%) patients (median duration 5 months, range 3-13), while the remaining 8/30 (27%) progressed. The increase in lymphocyte and eosinophil mean number was significantly higher in patients with PR or SD than in the progressed ones.(ABSTRACT TRUNCATED AT 250 WORDS)

Reversal of dorsoventral polarity in Xenopus laevis embryos by 180 degrees rotation of the animal micromeres at the eight-cell stage.

The 180 degrees rotation of the animal cell quartet at the 8-cell stage of Xenopus laevis embryos leads to a reversal of dorsoventral polarity in a majority of the embryos. Dorsal micromeres appear to instruct the macromeres in contact with them to initiate later steps in dorsal development. This inductive process is most apparent in the second half of the third cell cycle and is largely lost by the beginning of the fourth cell cycle.

Hemoglobin transition in the anuran Pelodytes punctatus.

In the anuran Pelodytes punctatus the larval hemoglobin produces five electrophoretic bands. In the premetamorphic period, two other bands appear which are typical of the adult. They gradually substitute the larval bands completely as in other anurans. The pathway of the hemoglobin shift indicates that the synthesis of the adult fractions does not depend on the thyroid hormones for its activation, whereas the disappearance of the larval fractions depends on the destruction of the "larval" red blood cell line. As in other species of anurans which are considered primitive, the hemoglobinic change of Pelodytes punctatus starts earlier and develops slower than in other more evolved anurans. In normal developing specimens of Pelodytes punctatus the change in the hemoglobin fractions occurs very slowly and finishes 4 weeks after the metamorphosis, whereas in "hibernated" specimens the rate of change is higher and it ends earlier, probably as a consequence in the retarded larvae, of the increased sensibility of the hemopoietic tissues to the metamorphosis factors.

Developmental time of the hemoglobin transition in the anuran Bombina orientalis.

The electrophoretic pattern of the larval hemoglobin of the anuran Bombina orientalis presents two bands. In premetamorphic period gradually appear four other bands, corresponding to those of the adult hemoglobin; they substitute, within 15-16 days after metamorphosis, the larval pattern. The percentage of larval and adult fractions during the development is shown. In Bombina orientalis the change of the hemoglobinic fractions from larval to adult type is total as in most anurans. But it starts earlier than in other species studied and develops slower than most of the other species.

Metamorphic variations in the hemoglobins of Bombina variegata (L.).

1. The electrophoretic pattern of the anuran Bombina variegata presents one band only until the end of premetamorphosis. Afterwards three new bands appear which substitute the larval band within 15-18 days of metamorphosis. 2. The gradual change of larval and adult hemoglobin, correlated with the development, has been reported in a diagram. 3. In Bombina variegata the hemoglobin change is total, like in most amphibians, but starts earlier and develops slower than in most other species.

Effects of fusion in pairs on the development of anurans Rana dalmatina and Rana esculenta.

Of 252 heterospecific, parabiotic pairs of Rana dalmatina and R. esculenta, fused during embryonic life, 87 died because the larva of R. dalmatina died at the beginning of its circulation. Later 89 pairs died when the R. esculenta larva died suddenly at the beginning of its independent feeding. Seventy-one pairs survived at the end of the metamorphic climax. Death in the first case is considered to result from some toxic effect, in the second from an immunological reaction. In the surviving pairs the duration of larval development in the rapidly developing species, i.e., R. dalmatina, was extended from two months to about four months, and in the more slowly developing species, i.e., R. esculenta, was reduced from about five months to about four. These changes in developmental rates are discussed in terms of changes in the concentration of the hormones controlling the rate of development due, in turn, to the parabiosis of embryos with different genetic constitution.

Teratogenic and toxic effects of alcohol ethoxylate and alcohol ethoxy sulfate surfactants on Xenopus laevis embryos and tadpoles.

The two surfactants tested, an alcohol ethoxylate (AE) and an alcohol ethoxy sulfate (AES), demonstrate both teratogenic and toxic effects in Xenopus laevis embryos and tadpoles. From acute tests and observations of malformations under light and electron microscopy, the AES produced less teratogenic and toxic effects than the AE, from which it differs only by the presence of a sulfate in the hydrophilic group of the molecule. The 72-h LC(50) was 4.59 mg/L for the AE and 6.75 g/L for the AES. The tissues most affected were the epithelia, particularly of the gills. As the AE exhibited ultrastructural alterations of mitochondria and narcotic effects, O(2) consumption was studied in treated tadpoles; results indicated collapse of the electrochemical gradient in mitochondria.

Tight junctions in early amphibian development: detection of junctional cingulin from the 2-cell stage and its localization at the boundary of distinct membrane domains in dividing blastomeres in low calcium.

Although functional studies indicate that tight junctions (TJ) are present in Xenopus laevis embryos from the 2-cell stage onward, morphological studies have failed to identify typical TJ structures before the 32-cell stage. Nothing is known about the expression and localization of TJ proteins in early Xenopus development. Here we have investigated the formation and composition of TJ in developing Xenopus embryos by whole-mount immunoperoxidase staining of eggs/embryos and immunoblotting of extracts with an antiserum against the TJ protein cingulin. Immunoblot analysis of eggs and embryo extracts showed that the antiserum labeled a major polypeptide of apparent M(r) 160 kD. Maternal cingulin was distributed diffusely in the cytocortical region of eggs and early embryos. Intense cingulin labeling became localized in the junctional region starting from the first cell division (2-cell stage). During cytokinesis, cingulin labeling was accumulated into new junctions in a precise spatial/temporal relationship with the deepening of the cleavage furrow. In semithin sections of stained embryos, labeling was detected in the most apical portion of the region of cell-cell contact. In embryos incubated in low calcium medium for 30 min, newly divided blastomeres failed to completely adhere to each other. However, cingulin labeling was accumulated along a linear structure that was at the border between distinct membrane domains (apical and lateral). These observations provide the first description of assembly of a TJ protein at the earliest stages of Xenopus development and suggest that TJ occur from the 2-cell stage onward and are assembled with maternal stores of protein. The formation of cingulin-containing structures even when lateral cell-cell adhesion is greatly reduced suggests that the apical cytocortex may have a determinative influence on TJ assembly and establishment of cell polarity.

Influence of reactive oxygen species production by monoamine oxidase activity on aluminum-induced mitochondrial permeability transition.

Treatment of Ca2+-loaded mitochondria with both aluminum and tyramine results in a swelling of higher amplitude than with aluminum alone, while tyramine alone is ineffective. The phenomenon is accompanied by H2O2 production and thiol and pyridine nucleotide oxidation. Cyclosporin A, N-ethylmaleimide or dithioerythritol completely prevent these effects, while catalase exhibits a lower inhibition, pointing to the induction of the permeability transition (MPT) by an oxidative stress. Reactive oxygen species are generated by the interaction of aluminum with the inner membrane and the oxidation of tyramine by monoamine oxidase on the outer membrane. This different localization determines the oxidation of critical thiol groups located on both internal and external sides of pore-forming structures, resulting in MPT induction. The reduced effect by aluminum or the inefficacy by tyramine, when implied alone, can be attributable to the oxidation of thiol groups located only on the internal or external side, respectively. Ultrastructural observations show that aluminum plus tyramine induce the typical configuration of mitochondria that have undergone the MPT. Instead, with aluminum alone, the sensitive subpopulation, although swollen, preserves the outer membrane and shows an apparently orthodox configuration.

Intercellular communication in the eight-cell stage of Xenopus laevis development: a study using dye coupling.

We report an investigation of dye coupling in the eight-cell stage of Xenopus laevis development. Our results indicate that fluorophors injected into micrometers at this stage pass only to sister cells (the corresponding macromeres) and that detectable dye transfer occurs only via cytoplasmic bridges, which persist for about the first two-thirds of the fourth cell cycle. We had previously shown that the dorsoventral polarity of the Xenopus embryo is regulated by a cell interaction that occurs at the end of the fourth cell cycle and we conclude that this cell interaction probably does not require cytoplasmic bridges or gap junctions.

Cadmium uptake and bioaccumulation in Xenopus laevis embryos at different developmental stages.

The uptake of cadmium in Xenopus laevis embryos was studied by exposing them to solutions containing cadmium concentrations ranging from 0.1 to 2 mg Cd2+/L at seven developmental stages for 72 h. The uptake values were from 0.0027 microgram Cd2+/embryo (two blastomeres stage) to 0.081 microgram Cd2+/embryo, (hindlimb bud distinct stage). During early developmental stages, a limited permeability to cadmium could play a significant role for the survival of embryos. At the last developmental stage evaluated there was a significantly higher uptake of cadmium simultaneously with a very significant increase in the resistance against cadmium toxicity. The cadmium bioaccumulation factor (BF) ranged from 5 to 460. As a general pattern at all developmental stages the BF was higher in embryos exposed to the lower cadmium concentration, and as development advanced the BF increased.

Globin evolution in some species of the genus Bufo.

The globins of 10 species of toads of the genus Bufo have been analysed. From the values of the reciprocal rates between their amino acid residues a 'dissimilarity matrix' was made. In this have been included the values related to Discoglossus pictus and Xenopus laevis. The numerical data, derived from a computer program, gave a dendrogram representing the evolution of the analysed globins. This appears correlated to the geographical distribution and to the metabolic adaptation of the corresponding species of Bufo more than their chronological appearance.