SURF1, encoding a factor involved in the biogenesis of cytochrome c oxidase, is mutated in Leigh syndrome

A drug with cationic characteristics such as procaine can be conveyed in a Carbomer hydrogel in two different ways: (i) in the form of salt in solution in the aqueous phase, and (ii) in the base form salified with the same polymer. Introduction of the drug into the hydrogel with different concentrations of polymer produced, in both cases, a reduction in viscosity in relation to drug concentration. The gels with procaine salified with the polymer showed greater viscosity. The drug release rate, in general, diminished with the increase in polymer concentration. Nevertheless, when this concentration was maintained, there was no variation in release rate when the viscosity produced as a consequence of drug concentration was changed. Gels with procaine salified with the carboxyvinylic polymer had a faster release rate than those with procaine in the hydrochloride form dissolved in the aqueous phase. These results have also been confirmed by a simulated absorption test.     

Ewing's sarcoma of the pelvis: changes over 25 years in treatment and results

The pelvic localisations of Ewing's sarcoma have the worst prognosis due to large size at diagnosis, frequent distant metastases, radiosensitive organs next to the tumour and difficult surgery. The purpose of the present study was to analyse treatment results over a period of 25 years and to investigate the impact of newer chemotherapy schedules, improved radiotherapy techniques and newer surgical methods on the prognosis. 35 children and young adults were identified from 1967 to 1994 for whom diagnosis, presentation, performed treatment and outcome were available. Tumour size, as measured from CT scans, response to chemotherapy and radiotherapy target volume, could be reviewed in the later years. Actuarial 5-year survival for the whole group was 31% and for the 24 non-metastatic patients 40%, with a disease-free interval of 19%. Tumour size could be measured in 27 patients and ranged from 36 to 1540 cm3. There were 12 local recurrences, 1 in the 4 patients treated with surgery. After 1983, 9 out of 17 irradiated patients developed local failure. 3 patients had adequate fields and one a close field which did not cover completely the prechemotherapy extent and 3 of these recurred. All 4 patients with stable disease after neoadjuvant CT failed locally, not withstanding high-dose radiotherapy. The mean length of neoadjuvant CT tended to be shorter in patients without local relapse. There was no significant difference in survival before and after 1983.     

Factor XI activation by meizothrombin: stimulation by phospholipid vesicles containing both phosphatidylserine and phosphatidylethanolamine

The activation of factor XI by meizothrombin was investigated using recombinant meizothrombin (R155A meizothrombin) that is resistant to autocatalytic removal of fragment 1. Meizothrombin was capable of activating factor XI at an activation rate similar to that of thrombin. Dextran sulphate and heparin, known cofactors of thrombin-mediated factor XI activation, did not stimulate the activation of factor XI by meizothrombin. However, the activation of factor XI by meizothrombin was markedly enhanced by vesicles containing phosphatidylcholine (PC), phosphatidylserine (PS) and phosphatidylethanolamine (PE), whereas PC/PS or PC/PE vesicles only had a minor effect on the activation. Thrombin-mediated factor XI activation was not influenced by phospholipids. The effect of PC/PS/PE and PC/PS vesicles was studied in a factor XI dependent clot lysis assay. In this assay, factor XI inhibits clot lysis by a feedback loop in the intrinsic pathway via thrombin-mediated factor XI activation. Removal of endogenous phospholipids in plasma by centrifugation resulted in an increased clot lysis, which could be restored to the pre-centrifugation level by the addition of PC/PS/PE vesicles, but not by PC/PS vesicles. When clot lysis was initiated by factor IXa in the presence of a factor XIa blocking antibody, there was no difference in inhibitory effect of PC/PS/PE or PC/PS vesicles. These data suggested that the differences in clot lysis inhibition observed between PC/PS/PE and PC/PS vesicles were caused by factor XI activation by meizothrombin. Meizothrombin-mediated factor XI activation may therefore play an important role in the antifibrinolytic feedback loop in the intrinsic pathway.     

A humanized form of a CD4-specific monoclonal antibody exhibits decreased antigenicity and prolonged plasma half-life in rhesus monkeys while retaining its unique biological and antiviral properties

Certain monoclonal antibodies (MAbs) directed against CD4 can efficiently block HIV-1 replication in vitro. To explore CD4-directed passive immunotherapy for prevention or treatment of AIDS virus infection, we previously examined the biological activity of a nondepleting CD4-specific murine MAb, mu5A8. This MAb, specific for domain 2 of CD4, blocks HIV-1 replication at a post-gp120-CD4 binding step. When administered to normal rhesus monkeys, all CD4+ target cells were coated with antibody, yet no cell clearance or measurable immunosuppression occurred. However, strong anti-mouse Ig responses rapidly developed in all monkeys. In the present study, we report a successfully humanized form of mu5A8 (hu5A8) that retains binding to both human and monkey CD4 and anti-AIDS virus activity. When administered intravenously to normal rhesus monkeys, hu5A8 bound to all target CD4+ cells without depletion and showed a significantly longer plasma half-life than mu5A8. Nevertheless, an anti-hu5A8 response directed predominantly against V region determinants did eventually appear within 2 to 4 weeks in most animals. However, when hu5A8 was administered to rhesus monkeys chronically infected with the simian immunodeficiency virus of macaques, anti-hu5A8 antibodies were not detected. Repeated administration of hu5A8 in these animals resulted in sustained plasma levels and CD4+ cell coating with humanized antibody for 6 weeks. These studies demonstrate the feasibility of chronic administration of CD4-specific MAb as a potential means of treating or preventing HIV-1 infection.     

Activity wheel running blunts increased plasma adrenocorticotrophin (ACTH) after footshock and cage-switch stress

N'-(3'-Monophospho-deoxyguanosin-8-yl)-N-acetylbenzidine (dGp-ABZ) is thought to play an important role in initiation of benzidine-induced bladder cancer in humans. This report assesses the possible formation of this adduct by peroxidatic activation of N-acetylbenzidine (ABZ). Adduct formation was measured by 32P-post-labeling. Ram seminal vesicle microsomes were used as a source of prostaglandin H synthase (PHS). The peroxidatic activity of PHS was compared with that for horseradish peroxidase. Both peroxidases converted ABZ to dGp-ABZ whether DNA or 2'-deoxyguanosine 3'-monophosphate (dGp) was present. Following 32P-post-labeling, the enzymatic and synthetic adduct were extracted from PEI-cellulose plates and were shown to have the same HPLC elution profiles for the bisphosphate adduct (32P-dpGp-ABZ). Treatment of the enzymatic and synthetic bisphosphate adduct with nuclease P1 yielded a product that eluted at the same time from the HPLC (32P-dpG-ABZ). Additional experiments demonstrated that the PHS-derived 5'-monophosphate (dpG-ABZ) and 3'-monophosphate (dGp-ABZ) adducts were also identical to their corresponding synthetic standard. With comparable amounts of total ABZ metabolism, PHS produced approximately 40-fold more dGp-ABZ than horseradish peroxidase (1943 +/- 339 versus 49 +/- 7.8 fmol/mg dGp). Adduct formation was dependent upon the presence of peroxidase and the specific substrate, i.e. arachidonic acid or H2O2. Adduct formation by PHS was inhibited by indomethacin (0.1 mM), ascorbic acid (1 mM) and glutathione (10 mM), but not by 5,5-dimethyl-1-pyrroline N-oxide (DMPO) (100 mM), a radical scavenger. Horseradish peroxidase adduct formation was also inhibited by ascorbic acid and glutathione. In addition, DMPO elicited greater than a 96% inhibition. Results demonstrate peroxidatic metabolism of ABZ to form dGp-ABZ. The mechanism of dGp-ABZ formation by PHS and horseradish peroxidase may be different.     

Ovariectomy-induced bone loss and the hematopoietic system

To investigate the relationship of the hematopoietic system to the loss of bone due to ovarian hormone deficiency, we examined the effects of ovariectomy and estrogen administration on the thymus, spleen and the bone marrow, and on the proliferation of marrow progenitors of osteoclasts. We also assessed the effects of daily administration of interleukin-1 receptor antagonist (IL-1ra) on bone loss due to ovarian hormone deficiency. Ovariectomy resulted in decreased cancellous bone volume, increased trabecular osteoblast and osteoclast numbers, and increased serum alkaline phosphatase levels that were prevented by 17 beta-estradiol treatment. Thymus weight, spleen weight, thymus and spleen lymphocytes, and bone marrow monocytes and lymphocytes also increased significantly following ovariectomy, and the increases were suppressed by 17 beta-estradiol. Ovariectomy, in addition, caused a 4-fold increase in the number of tartrate resistant acid phosphatase (TRAP)-positive multinucleated cells formed in cultures of marrow cells and the increase was partially inhibited by 17 beta-estradiol. IL-1ra administration did not prevent the bone loss due to ovariectomy. Our findings indicate that ovariectomy-induced bone loss in the rat is accompanied by marked changes in the hematopoietic system, and that these changes are modulated by estrogen administration. In spite of the negative finding with IL-1ra, the nature of the involvement of the hematopoietic system in the pathogenesis of bone loss due to ovarian hormone deficiency merits continued exploration.     

Regulation of mesangial cell ion channels by insulin and angiotensin II. Possible role in diabetic glomerular hyperfiltration

We used patch clamp methodology to investigate how glomerular mesangial cells (GMC) depolarize, thus stimulating voltage-dependent Ca2+ channels and GMC contraction. In rat GMC cultures grown in 100 mU/ml insulin, 12% of cell-attached patches contained a Ca(2+)-dependent, 4-picosiemens Cl- channel. Basal NPo (number of channels times open probability) was < 0.1 at resting membrane potential. Acute application of 1-100 nM angiotensin II (AII) or 0.25 microM thapsigargin (to release [Ca2+]i stores) increased NPo. In GMC grown without insulin, Cl- channels were rare (4%) and unresponsive to AII or thapsigargin in cell-attached patches, and less sensitive to [Ca2+]i in excised patches. GMC also contained 27-pS nonselective cation channels (NSCC) stimulated by AII, thapsigargin, or [Ca2+]i, but again only when insulin was present. In GMC grown without insulin, 15 min of insulin exposure increased NPo (insulin > or = 100 microU/ml) and restored AII and [Ca2+]i responsiveness (insulin > or = 1 microU/ml) to both Cl- and NSCC. GMC AII receptor binding studies showed a Bmax (binding sites) of 2.44 +/- 0.58 fmol/mg protein and a Kd (binding dissociation constant) of 3.02 +/- 2.01 nM in the absence of insulin. Bmax increased by 86% and Kd was unchanged after chronic (days) insulin exposure. In contrast, neither Kd nor Bmax was significantly affected by acute (15-min) exposure. Therefore, we concluded that: (a) rat GMC cultures contain Ca(2+)-dependent Cl- and NSCC, both stimulated by AII. (b) Cl- efflux and cation influx, respectively, would promote GMC depolarization, leading to voltage-dependent Ca2+ channel activation and GMC contraction. (c) Responsiveness of Cl- and NSCC to AII is dependent on insulin exposure; AII receptor density increases with chronic, but not acute insulin, and channel sensitivity to [Ca2+]i increases with both acute and chronic insulin. (d) Decreased GMC contractility may contribute to the glomerular hyperfiltration seen in insulinopenic or insulin-resistant diabetic patients.