Oxidation of N-hydroxyguanidines by cytochromes P450 and NO-synthases and formation of nitric oxide

Microsomal cytochromes P450 and tetrahydrobiopterin (BH4) free-NOS II catalyze the oxidation of N-hydroxyguanidines by NADPH and O2 with formation of nitrogen oxides including NO. These reactions are not selective in terms of substrates, as they occur on most N-hydroxyguanidines, and of products, as they not only lead to corresponding ureas but also to cyanamides. These non selective reactions are mainly due to O2- derived from the oxidase function of those hemeproteins. By contrast, NO synthase (NOS) containing BH4 catalyze the selective monooxygenation of some N-hydroxyguanidines by NADPH and O2 with formation of NO and the corresponding ureas in a 1:1 molar ratio. Those reactions are not inhibited by superoxide dismutase (SOD) and are performed by the NOS Fe(II)-O2 complex. The endogenous NOS substrate N(omega)-hydroxy-L-arginine (NOHA), and its close analogue homo-NOHA, are selectively oxidized in this manner by NOS whereas nor-NOHA and dinor-NOHA are not. Moreover, some non alpha-amino acid N-hydroxyguanidines act as NOS substrates in a manner similar to NOHA. This includes a small number of simple N-alkyl N'-hydroxyguanidines with R(alkyl) propyl, butyl, and pentyl, and some N-aryl N'-hydroxyguanidines that involve a relatively small and preferably electron-rich aryl substituent. The best exogenous substrate of NOS reported so far is N-butyl N'-hydroxyguanidine; this compound is oxidized by NOS II with formation of NO with a catalytic efficiency (kcat/Km) only two times lower than NOHA itself. N-butyl N'-hydroxyguanidine is also a good substrate for NOS I and NOS III. However, some N-aryl N'-hydroxyguanidines, with Ar = p-chlorophenyl and p-methylphenyl, are selective substrates of NOS II. These results show that exogenous N-hydroxyguanidines not bearing an alpha-amino acid function are efficiently and selectively oxidized by NOS with forrmation of NO. They open the way toward the research of new NO donors based on selective substrates of each class of NOS.     

Solid stress facilitates spheroid formation: potential involvement of hyaluronan

When neoplastic cells grow in confined spaces in vivo, they exert a finite force on the surrounding tissue resulting in the generation of solid stress. By growing multicellular spheroids in agarose gels of defined mechanical properties, we have recently shown that solid stress inhibits the growth of spheroids and that this growth-inhibiting stress ranges from 45 to 120 mmHg. Here we show that solid stress facilitates the formation of spheroids in the highly metastatic Dunning R3327 rat prostate carcinoma AT3.1 cells, which predominantly do not grow as spheroids in free suspension. The maximum size and the growth rate of the resulting spheroids decreased with increasing stress. Relieving solid stress by enzymatic digestion of gels resulted in gradual loss of spheroidal morphology in 8 days. In contrast, the low metastatic variant AT2.1 cells, which grow as spheroids in free suspension as well as in the gels, maintained their spheroidal morphology even after stress removal. Histological examination revealed that most cells in AT2.1 spheroids are in close apposition whereas a regular matrix separates the cells in the AT3.1 gel spheroids. Staining with the hyaluronan binding protein revealed that the matrix between AT3.1 cells in agarose contained hyaluronan, while AT3.1 cells had negligible or no hyaluronan when grown in free suspension. Hyaluronan was found to be present in both free suspensions and agarose gel spheroids of AT2.1. We suggest that cell-cell adhesion may be adequate for spheroid formation, whereas solid stress may be required to form spheroids when cell-matrix adhesion is predominant. These findings have significant implications for tumour growth, invasion and metastasis.     

A biomechanical study of two postoperative prostheses for transtibial amputees: a custom-molded and a prefabricated adjustable pneumatic prosthesis

We evaluated an adjustable pneumatic prefabricated prosthesis and a rigid custom-molded prosthesis for immediate postoperative use. Twelve transtibial amputations were performed on cadaver limbs. Differential variable reluctance transducers were placed subcutaneously across the wound edge medially and laterally. The limbs were then placed in either the pneumatic prosthesis (five limbs) or the rigid prosthesis (seven limbs). The specimens underwent static and cyclic loading to simulate weight bearing. The strain readings for static and cyclic loading were greater in the rigid prosthetic group. Only the mean medial strain measurement after cyclic loading was statistically significant. The results demonstrate that the pneumatic prosthesis places less strain across the wound than a rigid prosthesis.     

Interstitial fluid pressure in intracranial tumours in patients and in rodents.

Fluid transport parameters in intracranial tumours influence the delivery of therapeutic agents and the resolution of peritumoral oedema. The tumour and cortex interstitial fluid pressure (IFP) and the cerebrospinal fluid pressure (CSFP) were measured during the growth of brain and pial surface tumours [R3230AC mammary adenocarcinoma (R3230AC) and F98 glioma (F98)] in rats. Intratumoral and intracranial pressures were also measured in rodents and patients treated with dexamethasone, mannitol and furosemide (DMF), and hypocapnia. The results show that (1) for the R3230AC on the pial surface, IFP increased with tumour volume and CSFP increased exponentially for tumours occupying a brain volume of 5% or greater; (2) in F98 with volumes of approximately 10 mm3, IFP decreased from the tumour to the cortex, whereas for tumour volumes > 16 mm3 IFP equilibrates between F98 and the cortex; (3) DMF treatment reduced the IFP of intraparenchymal tumours significantly and induced a pressure gradient from the tumour to the cortex; and (4) in 11 patients with intracranial tumours, the mean IFP was 2.0 +/- 2.5 mmHg. In conclusion, the IFP gradient between intraparenchymal tumours and the cortex decreases with tumour growth, and treatment with DMF can increase the pressure difference between the tumour and surrounding brain. The results also suggest that antioedema therapy in patients with brain tumours is responsible in part for the low tumour IFP.     

Synergistic enhancement of herpes simplex virus thymidine kinase/ganciclovir-mediated cytoxicity by hydroxyurea

We have previously demonstrated (L. Z. Rubsam et al., Cancer Res., 59: 669-675, 1999) that low ganciclovir (GCV) triphosphate (TP) levels similar to cellular deoxynucleotide concentrations can induce multilog killing in cells stably expressing herpes simplex virus thymidine kinase (HSV-TK). In this study, we evaluated whether reducing the endogenous competitor of GCV-TP, dGTP, enhanced GCV-mediated cytotoxicity. In SW620 human colon carcinoma cells stably expressing HSV-TK, the addition of the ribonucleotide reductase inhibitor, hydroxyurea (HU), decreased cellular dGTP pools and simultaneously increased the accumulation of GCV-TP levels. The amount of GCV nucleotide transfer from HSV-TK-expressing to nonexpressing (bystander) cells was quantitated in physically separated pHook-expressing bystander cells. Elevation of the GCV-TP:dGTP ratio by HU resulted in increased levels of GCV nucleotides transferred from HSV-TK-expressing to bystander cells during a 24 h drug incubation and enhanced GCV monophosphate incorporation into DNA after drug removal. Isobologram analysis demonstrated that the combination of GCV and HU was additive in 100% HSV-TK cultures and synergistic in HSV-TK/bystander mixtures. IC50 values for GCV in 1:1 cocultures of HSV-TK-expressing and nonexpressing SW620 cells were reduced from 1.5 microM to 0.07 microM with 2 mM HU. A similar reduction was also observed with HT-29 cells and U251 cells. With 2 mM HU, IC50 values for GCV in 10:90, 5:95, and 1:99 SW620 HSV-TK-expressing and nonexpressing cocultures were reduced from 55 microM to 0.3 microM, 71 microM to 0.8 microM, and 118 microM to 7 microM, respectively. These results demonstrate the ability to pharmacologically enhance HSV-TK/GCV-mediated bystander killing and may have an important therapeutic impact.     

Effect of LY 171555 and CY 208-243 on tremor suppression in the MPTP monkey model of parkinsonism.

The antitremor effect of the D2 agonist LY 171555 and of the D1 agonist CY 208-243 alone and in combination was tested in a monkey previously rendered parkinsonian by MPTP and displaying exceptionally a rest tremor in the limbs. The D2 agonist suppressed rest tremor in a dose-dependent fashion. The D1 agonist by itself had no effect but it potentiated the effect of a small dose of LY 171555.     

Renin in hypertension: how important as a risk factor?

An analysis of the plasma renin levels in relation to the incidence of severe cardiovascular complications (coronary thrombosis, stroke, ruptured aortic aneurysm) was made in 325 patients with various types of hypertension. These patients had one to four measurements of plasma renin activity taken under standard conditions of sodium intake and posture in the period 1963-68. The follow-up was 5 to 10 years in the four groups of hypertensive patients (essential hypertension, malignant hypertension, hypertension secondary to renal parenchymatous disease and hypertension caused by, or associated with, renal artery obstruction). For all 325 patients, the incidence of such complications was 23.6, 20.4 and 44.7% in the low, normal and high renin groups. These findings are at variance with the claim that renin constitutes a serious risk factor in hypertensive patients, especially if it is isolated from other parameters such as the level of diastolic pressure, the adequacy of kidney function, the effectiveness of dietary and drug management of hypertension, and especially the presence or absence of atherosclerotic lesions of the large vessels at the time of the renin determination.