Alpha-oxidative metabolism of the bladder carcinogens N-nitrosobutyl(4-hydroxybutyl)amine and N-nitrosobutyl(3-carboxypropyl)amine within the rat isolated bladder

The most widely accepted metabolic pathway leading to the formation of reactive intermediates from nitrosamines involves enzymatic hydroxylation at the carbon atom alpha to the nitroso moiety. All subsequent steps are non-enzymatic reactions and the final result is the stoichiometric formation of a cationic product and molecular nitrogen. Thus the amount of molecular nitrogen evolved can be used as an indicator of alpha-hydroxylation. The use of doubly 15N-labelled nitrosamines and the detection of 15N2 by MS makes it simpler to measure the extent of alpha-hydroxylation. We have studied the alpha-oxidation of doubly 15N-labelled N-nitrosobutyl(4-hydroxybutyl)amine (BBN) and its metabolite N-nitrosobutyl(3-carboxypropyl)amine (BCPN), two potent urinary bladder carcinogens in animals, within the target organ. Various amounts of 15N-labelled BBN ranging from 0.1 to 5 mumol were incubated at 37 degrees C for 4 h in the isolated rat bladder and the formation of 15N2 was measured by GC-MS. 15N2 production was linear up to 1 mumol and represented approximately 0.1% of the substrate incubated. Time-course experiments showed that 15N2 production was linear over a 6 h incubation period, ranging from 2.16 +/- 0.05 to 4.55 +/- 0.33 nmol/mg urothelial cell protein. 15N-labelled BCPN (1-5 mumol) was also incubated within the rat isolated bladder. 15N2 production from BCPN was approximately 10 times less than that from BBN. The results indicate that, though to a lower extent, the target organ activates 15N-labelled BBN and BCPN through the alpha-hydroxylation pathway.     

Radial Glia-Like Cells in the Supraoptic Nucleus of the Adult Rat

Conventional light and confocal microscopy of thick vibratome sections of the hypothalamus of adult male and female rats immunostained for the astrocytic marker glial fibrillary acidic protein (GFAP) revealed that the supraoptic nucleus (SON) contains two morphologically distinct types of astrocytes. One has a stellate form, similar to that of most astrocytes in the adult CMS. The other has a morphology reminiscent of radial glia in the developing CNS: from their cell bodies, located along the ventral glia lamina (VGL), arise one long thick process that spans the SON in the coronal plane, several horizontally-oriented processes that form a dense network in the VGL, and a short process oriented towards the pia. The latter astrocytes are immunoreactive for vimentin, an intermediate filament protein of immature glial cells and a marker for radial glia. The stellate astrocytes showed no vimentin immunoreactivity. The functional significance of each type of supraoptic astrocyte is at present unknown but the presence of radial glia-like cells in this hypothalamic region suggests that the SON retains a certain degree of immaturity during adulthood, that may be linked to its well known capacity to undergo neuronal-glial plasticity under physiological and experimental stimulation.     

Pharmacokinetic profile and metabolism of N-nitrosobutyl-(4-hydroxybutyl)amine in rats

N-Nitrosodibutylamine and its omega-hydroxylated metabolite N-nitrosobutyl(4-hydroxybutyl)amine (NB4HBA) induce tumors in the urine bladder of different animal species through their common urinary metabolite N-nitrosobutyl(3-carboxypropyl)amine (NB3CPA), resulting from the oxidation of the alcoholic group of NB4HBA to a carboxylic group. NB4HBA disappearance from blood, the formation of its main metabolites, NB3CPA and NB4HBA-glucuronide (NB4HBA-G), and their urinary excretion, were investigated in rats after an i.v. dose of 1 mg/kg (5.7 mumol/kg). NB3CPA and NB4HBA-G formation was readily detectable 2 min after treatment and levels were still measurable at 120 and 30 min, respectively. The parent compound disappeared from blood 90 min after injection. The NB4HBA blood concentration-time profile was adequately described by a one-compartmental linear model. NB4HBA half-life was 8 min, total body clearance and renal clearance were 86.1 and 0.22 ml/min/kg, respectively. The 0-96-h urinary excretion of NB4HBA was 0.3% of the administered dose. NB3CPA half-life was 15 min; NB3CPA and NB4HBA-G urinary excretion were 36 and 11.7%, respectively, urinary excretion of known compounds accounting for less than 50%. After i.v. injection of NB3CPA equimolar to the NB4HBA dose, only 50% of unchanged compound was recovered in the urine and after NB4HBA-G, 41% of the administered dose was excreted unchanged, NB3CPA accounting for 10%. Thus NB3CPA and NB4HBA-G might undergo further biotransformation, suggesting that NB3CPA may not be the ultimate carcinogen responsible for urinary bladder tumor induction.     

Differential desensitization of ionotropic non-NMDA receptors having distinct neuronal location and function

The release of tritium from rat hippocampal synaptosomes prelabeled with [³H]noradrenaline ([³H]NA) or [³H]5-hydroxytryptamine ([³H]5-HT) and from rat neocortex synaptosomes prelabeled with [³H]choline and the release of endogenous GABA and glutamate from rat neocortex synaptosomes were monitored during superfusion with media containing varying concentrations of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) or kainic acid. Concentration-dependent release potentiations were elicited by both excitatory amino acids (EAAs) in all the transmitter systems investigated. The releases evoked by 100 μM AMPA were, in all cases, almost totally dependent on external Ca²⁺ and sensitive to 6,7-dinitroquinoxaline-2,3-dione (DNQX), indicating involvement of non-NMDA receptors. When cyclothiazide, a drug able to prevent desensitization of AMPA-preferring receptors, was added to the superfusion medium (at 1 or 10 μM) concomitantly with 100 μM AMPA or kainate, the EAA-evoked release of [³H]NA was significantly enhanced. Concanavalin A, a lectin thought to prevent desensitization of kainate-preferring receptors, had no effect (up to 10 μM) on the release of [³H]NA evoked by AMPA or kainate. The effect of cyclothiazide was lost if, after an 8-min pretreatment, the drug was removed just before the AMPA stimulus. When added concomitantly with the EAAs, cyclothiazide potentiated the release of [³H]5-HT elicited by AMPA and, less so, that evoked by kainate. Concanavalin A was ineffective. Neither cyclothiazide (1 or 10 μM) nor concanavalin A (3 or 10 μM) could affect the release of [³H]ACh or endogenous GABA provoked by 100 μM AMPA or kainate, suggesting that the receptors involved do not desensitize. Exposure of neocortex synaptosomes to AMPA or kainate concomitantly with cyclothiazide caused endogenous glutamate release that did not differ from that evoked by the EAAs alone. In contrast, the effects of AMPA and kainate were potentiated by concanavalin A. The activity of the lectin (3 μM) persisted when it was applied for 8 min and then removed before the AMPA or kainate (100 μM) pulse. When hippocampal synaptosomes prelabeled with [³H]NA were subjected to three subsequent AMPA (100 μM) stimuli (S₁, S₂ and S₃), the release of [³H]NA decreased dramatically from S₁ to S₃ (S₃/S₁ = 0.14 ± 0.04); a significant ‘protection’ of the AMPA effect was offered by 1 μM cyclothiazide (S₃/S₁ = 0.36 ± 0.06). This value did not differ from the S₃/S₁ ratio (0.38 ± 0.04) obtained in parallel experiments with 12 mM K⁺. The release evoked by high-K⁺ was insensitive to cyclothiazide. Finally, the effect of AMPA on the release of [³H]ACh did not respond to cyclothiazide also during three subsequent stimuli with 100 μM AMPA. To conclude: a) ionotropic non-NMDA receptors mediating enhancement of NA, 5-HT, ACh, GABA and glutamate release exist on the corresponding nerve terminals; b) the receptors present on noradrenergic and serotonergic neurons are AMPA-preferring receptors, whereas the glutamate autoreceptors resemble most the kainate-preferring subtype; the receptors mediating ACh and GABA release can not be subclassified at present; c) desensitization may not be a property of all non-NMDA ionotropic receptors. The receptors here characterized represent five models of native non-NMDA receptors suitable for pharmacological and molecular studies. Received: 28 January 1997 / Accepted: 14 April 1997     

Early and late results of extended surgery for cancer of the stomach

The experience of the Istituto Nazionale Tumori of Milan of 143 patients who underwent extended surgery for cancer of the stomach from 1965 to 1980 is reviewed. They represent 16.3 per cent of the patients who underwent curative surgery. The operative mortality rate was 15.4 per cent but this significantly decreased in recent years to 8 per cent and the morbidity rate to 17.5 per cent. The overall 5-year survival rate was 19 per cent. Survival was analysed according to tumour penetration (pT) and nodal status (N). It was found that patients without tumour penetration of adjacent structures and nodal involvement (pT3N-) had a better 5-year survival rate (21 per cent) than patients with nodal involvement (pT3N+) (2 per cent). Patients with tumour penetration of adjacent structures and without nodal involvement (pT4N-) had a better 5-year survival rate (29 per cent) than patients with nodal involvement (pT4N+) (5 per cent). These differences were significant on log rank test (P less than 0.000001 and P less than 0.001 respectively) and suggest that nodal status is a stronger prognostic variable than pT level. The role of extended surgery is discussed from the viewpoint of the oncological surgeon who has to weigh up the difficulty of a preoperative diagnosis of tumour infiltration of adjacent structures (predictive positive value 0.39), with the operative mortality rate of at least 8 per cent and long-term results which are strongly affected by the nodal status.     

Osteopenia and osteoporosis in HIV+ patients, untreated or receiving HAART.

In the last few years there are increasing evidences suggesting that osteopenia and osteoporosis are frequent among HIV positive patients. It is still not clear if the bone demineralization is a direct consequence of viral infection or of the antiretroviral drugs. Studies to date therefore give conflicting results. We performed a study to evaluate the prevalence of osteopenia and osteoporosis in HIV positive patients, either untreated or receiving antiretroviral therapy, to asses the frequency of these conditions and the role of antiretroviral drugs.     

Arachnoid cysts: diagnosis and treatment

Twenty supratentorial and 10 infratentorial arachnoid cysts are reported. The patients were from 0 to 15 years of age. The commonest presenting symptoms in children were cranial enlargement, epileptic seizures, and psychomotor retardation. Neuroradiological evaluation included CT, metrizamide CT, cisternography, and angiography. Echography was performed in 5 newborns. Therapeutic criteria according to the clinical and neuroradiological findings are reviewed. Cystoperitoneal shunting in combination with ventriculoperitoneal shunting for associated hydrocephalus is considered the treatment of choice.     

Chloroquine‐treated dendritic cells require STAT1 signaling for their tolerogenic activity

MS and EAE are T cell‐driven autoimmune diseases of the CNS where IL‐17‐producing Th17 cells promote damage and are pathogenic. Conversely, tolerogenic DCs induce Treg cells and suppress Th17 cells. Chloroquine (CQ) suppresses EAE through the modulation of DCs by unknown mechanisms. Here, we show that STAT 1 is necessary for CQ‐induced tolerogenic DCs (tolDCs) to efficiently suppress EAE. We observed that CQ induces phosphorylation of STAT1 in DCs in vivo and in vitro. Genetic blockage of STAT1 abrogated the suppressive activity of CQ‐treated DCs. Opposed to its WT counterparts, CQ‐treated STAT1^?/? BMDCs were unable to suppress Th17 cells and increased EAE severity. Our findings show that STAT1 is a major signaling pathway in CQ‐induced tolDCs and may shed light on new therapeutic avenues for the induction of tolDCs in autoimmune diseases such as MS.     

No evidence for genetically determined alteration in insulin secretion or sensitivity predisposing to type 1 diabetes: a study of identical twins

OBJECTIVE: To determine whether inherited changes in insulin secretion or sensitivity could predispose to type 1 diabetes, we studied identical twins of type 1 diabetic patients. RESEARCH DESIGN AND METHODS: We studied prospectively a consecutive series of 27 identical twins of patients with type 1 diabetes who were initially nondiabetic, as well as 14 control subjects, over a period of 18 years. Of these 27 twins, 15 remain nondiabetic (now estimated at low disease risk) and 12 developed diabetes (pre-diabetic twins). Subjects were tested when not diabetic on at least two occasions with an intravenous glucose tolerance test (IVGTT), and we estimated insulin secretion as first-phase insulin response (FPIR), glucose clearance (K(g)), and insulin sensitivity both by homeostasis model assessment of insulin resistance (HOMA-IR) and relative to insulin response by the basal HOMA-IR-to-FPIR ratio. RESULTS: Twins now at low risk and control subjects had similar fasting blood glucose and insulin levels, FPIR, K(g), HOMA-IR, and HOMA-IR-to-FPIR ratio. In contrast, pre-diabetic twins compared with control twins had higher fasting insulin levels (10.3 +/- 6.0 vs. 4.6 +/- 4.0 mIU/ml), lower FPIR (245 +/- 129 vs. 796 +/- 622 mIU . ml(-1) . 10 min(-1)), lower K(g) (1.5 +/- 0.6 vs. 2.6 +/- 0.8% per min), and higher HOMA-IR-to-FPIR ratio (0.007 +/- 0.005 vs. 0.001 +/- 0.0009) (all P < 0.01). CONCLUSIONS: These observations in low-risk nondiabetic identical twins failed to identify a familial alteration in either insulin secretion or sensitivity predisposing to type 1 diabetes.