SUPPRESSION OF PIGMENTATION IN MOUSE MELANOMA CELLS BY 5-BROMODEOXYURIDINE : Effects on Tyrosinase Activity and Melanosome Formation

Low concentrations (1–3 µg/ml) of 5-bromodeoxyuridine (BrdU) reversibly suppress pigmentation in a highly pigmented clone (B559) of cultured B16 mouse melanoma cells. We have found that unpigmented cells (clone C3471), derived by long-term culture of B559 cells in 1 µg of BrdU/ml, were completely amelanotic with no biochemically or cytochemically detectable tyrosinase activity or ultrastructural evidence of premelanosomes. The process by which pigmentation is suppressed was studied in B559 cells during a 7-day period of growth with BrdU (3 µg/ml). Assays of tyrosinase activity showed that activity was reduced after 1 day and decreased progressively, approaching zero by 7 days. A quantitatively minor part of this reduction was directly attributable to the appearance of a dialyzable inhibitor of tyrosinase activity. Acrylamide gel electrophoresis revealed two bands of activity corresponding in Rx values to the T1 and T2 forms of soluble tyrosinase. Both were progressively reduced during growth with BrdU but one form (T1) was consistently affected earlier than the other (T2). Ultrastructural-cytochemical studies also showed an early effect on the localization of tyrosinase reaction product. At day 3, reaction product was no longer present in Golgi saccules and Golgi-associated smooth surfaced tubules, but was still seen within premelanosomes, compound melanosomes, and occasional Golgi-associated vesicles. By 7 days tyrosinase reaction product was usually not demonstrable. The number of premelanosomes was progressively decreased during growth with BrdU. Premelanosomes became concentrated in the juxtanuclear region and at day 3 many were contained within abnormally large and numerous compound melanosomes. Premelanosomes and compound melanosomes were rarely seen at 7 days, by which time the cultures were nearly amelanotic. The coordinated suppression of melanogenesis by BrdU may provide a useful model in which to study the normal regulation of this process

Hyperoxemia and excess oxygen use in early acute respiratory distress syndrome : Insights from the LUNG SAFE study

Publisher Copyright: © 2020 The Author(s). Copyright: Copyright 2020 Elsevier B.V., All rights reserved.Background: Concerns exist regarding the prevalence and impact of unnecessary oxygen use in patients with acute respiratory distress syndrome (ARDS). We examined this issue in patients with ARDS enrolled in the Large observational study to UNderstand the Global impact of Severe Acute respiratory FailurE (LUNG SAFE) study. Methods: In this secondary analysis of the LUNG SAFE study, we wished to determine the prevalence and the outcomes associated with hyperoxemia on day 1, sustained hyperoxemia, and excessive oxygen use in patients with early ARDS. Patients who fulfilled criteria of ARDS on day 1 and day 2 of acute hypoxemic respiratory failure were categorized based on the presence of hyperoxemia (PaO2 > 100 mmHg) on day 1, sustained (i.e., present on day 1 and day 2) hyperoxemia, or excessive oxygen use (FIO2 ≥ 0.60 during hyperoxemia). Results: Of 2005 patients that met the inclusion criteria, 131 (6.5%) were hypoxemic (PaO2 < 55 mmHg), 607 (30%) had hyperoxemia on day 1, and 250 (12%) had sustained hyperoxemia. Excess FIO2 use occurred in 400 (66%) out of 607 patients with hyperoxemia. Excess FIO2 use decreased from day 1 to day 2 of ARDS, with most hyperoxemic patients on day 2 receiving relatively low FIO2. Multivariate analyses found no independent relationship between day 1 hyperoxemia, sustained hyperoxemia, or excess FIO2 use and adverse clinical outcomes. Mortality was 42% in patients with excess FIO2 use, compared to 39% in a propensity-matched sample of normoxemic (PaO2 55-100 mmHg) patients (P = 0.47). Conclusions: Hyperoxemia and excess oxygen use are both prevalent in early ARDS but are most often non-sustained. No relationship was found between hyperoxemia or excessive oxygen use and patient outcome in this cohort. Trial registration: LUNG-SAFE is registered with ClinicalTrials.gov, NCT02010073publishersversionPeer reviewe

Altered Glutamate Receptor Function during Recovery of Bladder Detrusor-External Urethral Sphincter Coordination in a Rat Model of Spinal Cord Injury

ABSTRACT Coordination of the bladder detrusor and the external urethral sphincter is a supraspinally controlled reflex that is essential for efficient micturition. This coordination is permanently lost after spinal cord transection but can recover chronically after incomplete spinal cord injury (SCI). As glutamatergic transmission plays a key role in all levels of detrusor-external urethral sphincter coordination, we examined the role of potential alterations in glutamatergic control in its recovery after SCI. Rats were subjected to standardized incomplete contusion injury. Detrusorexternal urethral sphincter coordination was evaluated urodynamically at 5 days (subacute) and 8 weeks (chronic) after SCI. Sensitivity of coordinated activation of the external urethral sphincter in response to bladder distension to the ␣ -amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid/kainate antagonist 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo(f)quinoxaline-7-sulfonamide disodium (NBQX) and to the N-methyl-Daspartate (NMDA) antagonist R(Ϫ-3-(2-carboxypiperazine-4-yl)-propyl-1-phosphonic acid (CPP) was determined by intrathecal application at the L6 spinal cord level during urodynamic recordings. We found that while detrusor contractions recovered at 5 days after SCI, coordinated activation of the external urethral sphincter was significantly impaired at 5 days and recovered only by 8 weeks. There was no difference in sensitivity of detrusor-external urethral sphincter coordination to NBQX at the subacute or chronic time points. However, external urethral sphincter response to bladder distension was sensitive to a 50% lower dose of CPP at 5 days compared with uninjured rats or chronic recovered SCI rats. Thus, alterations in NMDA receptor function appeared to be involved in recovery of detrusor-external urethral sphincter coordination after incomplete SCI

Stem Cells in Spinal Cord Injury

Traumatic injury to the adult spinal cord results in a massive loss of cells and permanent functional deficits. However, recent studies demonstrate that there is a proliferative response of endogenous glial precursors and progenitors and perhaps also pluripotent neural stem cells. These cells may prove to be an important new therapeutic target to improve recovery after injury to the spinal cord and brain

Up-regulation of 5-HT2 receptors is involved in the increased H-reflex amplitude after contusive spinal cord injury

The amplitude of the H-reflex increases chronically after incomplete SCI and is associated with the development of exaggerated hindlimb reflexes. Although the mechanism for this increased H-reflex is not clear, previous studies have shown that pharmacological activation of the 5-HT(2) receptors (5-HT(2)R) can potentiate the monosynaptic reflex. This study tested the hypothesis that increased expression of 5-HT(2)R on motoneurons is involved in increased H-reflex amplitude after a standardized clinically-relevant contusive SCI. Adult female rats were subjected to contusion, complete surgical transection, or a T8 laminectomy only. At 4wks after surgery, H-reflex recordings from the hindpaw plantar muscles of contused rats showed twice the amplitude of that in laminectomy controls or transected rats. To probe the role of 5-HT(2)R in this increased amplitude, dose response studies were done with the selective antagonists, mianserin or LY53857, and the 5-HT(2)R agonist, (±)-1-(2,5-Dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride (DOI). The drugs were intrathecally infused into the lumbar cord while recording the H-reflex. Mianserin did not have any significant effects on the H-reflex after transection, consistent with the loss of distal serotonergic innervation. After contusion, both 5-HT(2)R antagonists reduced the H-reflex reflex amplitude with a significantly higher ID(50) compared to the uninjured controls. The 5-HT(2)R agonist, DOI, significantly increased reflex amplitude in contused but not control rats. Furthermore, while 5-HT immunoreactivity was similar, contused rats displayed increased 5-HT(2A)R immunoreactivity in plantar muscle motoneurons compared to uninjured controls. We conclude that increased expression of 5-HT(2)R is likely to be involved in the enhanced H-reflex that develops after contusive SCI