The effect of tryptophan plus methionine, 5-azacytidine, and methotrexate on adjuvant arthritis of rat.

Within the wider framework of our studies on the genesis of rheumatoid arthritis we have investigated the two signal processes in arthritis: adenoribosylation of proteins and DNA methylation. Arthritis can be induced when Freund's complete adjuvant is applied to rats. This form of arthritis can then be reduced or even totally suppressed through the application of several different substances. In the present article we have investigated if the effect of two of these substances, 5-azacytidine and methotrexate can be influenced by the application of tryptophan plus methionine. When applied singly, these latter two substances are known to reduce the formation of arthritis. This effect is intensified by a combination of tryptophan plus methionine. Application of tryptophan plus methionine without methotrexate or 5-azacytidine causes an enhanced development of an adjuvant induced arthritis.     

Quantification of longitudinal tissue pO2 gradients in window chamber tumours: impact on tumour hypoxia

We previously reported that the arteriolar input in window chamber tumours is limited in number and is constrained to enter the tumour from one surface, and that the pO2 of tumour arterioles is lower than in comparable arterioles of normal tissues. On average, the vascular pO2 in vessels of the upper surface of these tumours is lower than the pO2 of vessels on the fascial side, suggesting that there may be steep vascular longitudinal gradients (defined as the decline in vascular pO2 along the afferent path of blood flow) that contribute to vascular hypoxia on the upper surface of the tumours. However, we have not previously measured tissue pO2 on both surfaces of these chambers in the same tumour. In this report, we investigated the hypothesis that the anatomical constraint of arteriolar supply from one side of the tumour results in longitudinal gradients in pO2 sufficient in magnitude to create vascular hypoxia in tumours grown in dorsal flap window chambers. Fischer-344 rats had dorsal flap window chambers implanted in the skin fold with simultaneous transplantation of the R3230AC tumour. Tumours were studied at 9-11 days after transplantation, at a diameter of 3-4 mm; the tissue thickness was 200 microm. For magnetic resonance microscopic imaging, gadolinium DTPA bovine serum albumin (BSA-DTPA-Gd) complex was injected i.v., followed by fixation in 10% formalin and removal from the animal. The sample was imaged at 9.4 T, yielding voxel sizes of 40 microm. Intravital microscopy was used to visualize the position and number of arterioles entering window chamber tumour preparations. Phosphorescence life time imaging (PLI) was used to measure vascular pO2. Blue and green light excitations of the upper and lower surfaces of window chambers were made (penetration depth of light approximately 50 vs >200 microm respectively). Arteriolar input into window chamber tumours was limited to 1 or 2 vessels, and appeared to be constrained to the fascial surface upon which the tumour grows. PLI of the tumour surface indicated greater hypoxia with blue compared with green light excitation (P < 0.03 for 10th and 25th percentiles and for per cent pixels < 10 mmHg). In contrast, illumination of the fascial surface with blue light indicated less hypoxia compared with illumination of the tumour surface (P < 0.05 for 10th and 25th percentiles and for per cent pixels < 10 mmHg). There was no significant difference in pO2 distributions for blue and green light excitation from the fascial surface nor for green light excitation when viewed from either surface. The PLI data demonstrates that the upper surface of the tumour is more hypoxic because blue light excitation yields lower pO2 values than green light excitation. This is further verified in the subset of chambers in which blue light excitation of the fascial surface showed higher pO2 distributions compared with the tumour surface. These results suggest that there are steep longitudinal gradients in vascular pO2 in this tumour model that are created by the limited number and orientation of the arterioles. This contributes to tumour hypoxia. Arteriolar supply is often limited in other tumours as well, suggesting that this may represent another cause for tumour hypoxia. This report is the first direct demonstration that longitudinal oxygen gradients actually lead to hypoxia in tumours.     

Biological Characteristics of Micrometastatic Cancer Cells in Bone Marrow

There is emerging evidence that epithelial tumor cells are able to disseminate to secondary organs at an early stage of primary tumor development. One of the most prominent secondary organs screened for this type of dissemination is bone marrow. Even in cancer entities where overt skeletal metastases are rare (e.g., colorectal and ovarian cancer), bone marrow is a prognostically relevant indicator organ for the presence of hematogenous micrometastases. The currently available data suggest that bone marrow micrometastases represent a selected population of dormant cancer cells which still express a considerable degree of heterogeneity. The analysis of micrometastatic cells will open a new avenue to assess the molecular determinants of early tumor cell dissemination and subsequent outgrowth into overt metastases. Moreover, monitoring the elimination of bone marrow micrometastases and identification of treatment-resistant tumor cell clones may help to increase the efficacy of adjuvant therapy. This review summarizes the current knowledge on the biological characteristics of micrometastatic cancer cells in bone marrow of patients with solid epithelial malignancies.     

Toxicity of the cyanobacterial cyclic heptapeptide toxins microcystin-LR and -RR in early life-stages of the African clawed frog (Xenopus laevis)

Numerous cyanobacterial species are capable of producing potent toxins, which have been known to cause intoxications and fatalities in wildlife, livestock and humans. Microcystis is amongst the most ubiquitously distributed blue-green algal genus and almost invariably produces cyclic heptapeptide toxins called microcystins (MC). These toxins are highly persistent in water (several weeks). Highest concentrations are found in shallow littoral areas, the primary environment for aquatic early life-stage development. Therefore, the present study focussed on the potential embryotoxic effects of MC (MC-LR and -RR) in early life-stages of the amphibian Xenopus laevis. The endpoints chosen were mortality, malformation and growth inhibition. To achieve an improved dose-response relationship the uptake of MC was quantified simultaneously, using a radiolabeled derivative of MC-LR. As one of the best described molecular mechanisms of MC toxicity involves the specific inhibition of serine/threonine protein phosphatases-1 and -2A (PP), essential enzymes involved in the mechanisms of cell cycle regulation and maintenance of cellular morphology, the inhibition of PP in X. laevis exposed to MC was monitored. For this the presence of both PP-1 and PP-2A was confirmed by means of SDS-PAGE and immunoblotting. Second, the capacity of MC to inhibit X. laevis embryo-larval PP was corroborated by in vitro incubation of embryo-larval homogenates with MC-LR and -RR and subsequent determination of PP-inhibition. No increased mortality, malformation, or growth inhibition was observed even at the highest MC concentrations employed. MC had neither a demonstrable inhibitory effect on X. laevis PP-activity in vivo in the first 96 h of exposure. However, as of 96 and 120 h exposure a significant inhibition of PP activity was observed at the highest dose (2000 μg/l) in MC-LR and MC-RR exposed embryo-larvae, respectively. By the same token, no notable amounts of radiolabeled [3H]-MC-LR were taken up during the first 96 h, whereas a drastic increase in [3H]-MC-LR was observed after feeding of the larvae had commenced. The [3H]-MC-LR concentration was consistently found to be highest in the viscerothoracal sections of the larvae (2112+/-429 μg MC/kg dry weight after 120 h). The present findings indicate that transchorional/transdermal absorption of MC in X. laevis is minimal or absent and that oral uptake of MC with ambient water is necessary for the development of MC related toxicity. Furthermore, the comparison of the MC doses used in this study with the concentrations reported in surface waters indicate that early life-stages of amphibians (up to 5 days of development) are unlikely to be affected by cyanobacterial blooms producing MC-LR and -RR.     

Der einflu\ der entfernung auf das richtungshören

Zusammenfassung Beim Richtungshören ist jedem Laufzeitunterschied eine bestimmte Schallrichtungsempfindung zugeordnet. Wie aber verschiedene Messungen und Berechnungen gezeigt haben, verändert sich der Zeitunterschied mit dem Richtungswinkel nicht direkt proportional, sondern der Anstieg der Zeitdifferenz wird mit größeren Schalleinfallswinkeln zunehmend kleiner. Trotzdem stimmt normalerweise bei nicht zu kleiner Prüfentfernung der subjektive Richtungswinkel mit dem. objektiven Richtungswinkel überein. Abweichungen treten nur dann auf, wenn die Schallquelle sehr klein ist and sich verhältnismäi ßig nahe am Kopf des Beobachters befindet. Daß in solchen Fällen Lokalisationsfehler unvermeidlich sind, konnte durch eingehende Berechnungen gezeigt werden. Auf diese Weise läßt sich auch die von Jongkees u. v. n. Veer gefundene S-Kurve des Richtungshörens eindeutig erklären.     

The renoprotective potential of endothelin receptor antagonists

The endothelin system has been identified as having a substantial role in renal failure, both acute and chronic. Beside its well characterised haemodynamic effects, its mitogenic and pro-fibrotic properties have gained increased interest in the pathophysiology of chronic renal failure. This review outlines the role of endothelin in the pathogenesis of various renal diseases with a special focus on the potential of blocking this system with endothelin receptor antagonists. So far, most data were derived from animal models, but they provide strong evidence that endothelin receptor antagonists may represent a powerful therapeutic strategy in ameliorating the course of acute and chronic renal failure.     

Review of methods used to study oxygen transport at the microcirculatory level

The existence of hypoxic regions in tumors has long been recognized as a key factor leading to radiation resistance. More recently, it has been found that low oxygen levels also affect drug resistance, angiogenesis, cytokine production, cell cycle control, apoptosis, and metastatic propensity of tumors. Until now, most approaches to eliminating hypoxia have been empirical. However, improved understanding of the underlying mechanisms may permit the development of more soundly based, effective approaches. As discussed in this review, critical evaluation of the factors governing oxygen transport in tumors requires a thorough understanding of the methods used to study this process. Many experimental methodologies can be used to address these issues. In this review, the emphasis is placed on techniques that measure parameters on the scale of the diffusion distance of oxygen. Studies at the microregional level provide the most detailed physiological information on such processes. Over the past few years, significant progress in technology has allowed us to measure tumor oxygenation, yet spatial and temporal heterogeneities continue to provide significant challenges to obtaining clear knowledge of oxygen transport.     

Initial stages of tumor cell-induced angiogenesis: evaluation via skin window chambers in rodent models

BACKGROUND: There is a paucity of information about events that follow immediately after tumor cells are triggered to initiate the process of angiogenesis (the formation of new blood vessels). Such information is relevant to the issue of when micrometastases vascularize and has implications for the accessibility of micrometastases to various treatments. In this study, we attempted to monitor events at the initiation of angiogenesis at the earliest possible stage of tumor growth in vivo. METHODS: Two different rodent mammary tumor cell lines, R3230Ac from the Fischer 344 rat and 4T1 from the BALB/c mouse, were stably transfected with a gene that encodes an enhanced version of green fluorescence protein (GFP). GFP-labeled R3230Ac or 4T1 cells (about 20-50 cells) were implanted into dorsal skinfold window chambers of Fischer 344 rats or BALB/c mice, respectively. Tumor angiogenesis was then monitored serially and noninvasively for up to 4 weeks. RESULTS: Clear evidence of modification of the host vasculature was observed when tumor mass reached approximately 60-80 cells, and functional new blood vessels were seen when tumor mass reached roughly 100-300 cells. Individual tumor cells exhibited a chemotaxis-like growth pattern toward the pre-existing host vasculature. When ex-flk1 (a soluble, truncated vascular endothelial cell growth factor receptor protein known to be antiangiogenic) was injected with the tumor cells, the initial angiogenic and tumor growth activities were inhibited considerably, indicating that angiogenesis inhibitors may halt tumor growth even before the onset of angiogenesis. CONCLUSION: Angiogenesis induced by tumor cells after implantation in the host begins at a very early stage, i.e., when the tumor mass contains roughly 100-300 cells. Identification of chemotactic signals that initiate tumor cell migration toward the existing vasculature may provide valuable targets for preventing tumor progression and/or metastases.