The ATP/DNA ratio is a better indicator of islet cell viability than the ADP/ATP ratio.

Real-time, accurate assessment of islet viability is critical for avoiding transplantation of nontherapeutic preparations. Measurements of the intracellular ADP/ATP ratio have been recently proposed as useful prospective estimates of islet cell viability and potency. However, dead cells may be rapidly depleted of both ATP and ADP, which would render the ratio incapable of accounting for dead cells. Since the DNA of dead cells is expected to remain stable over prolonged periods of time (days), we hypothesized that use of the ATP/DNA ratio would take into account dead cells and may be a better indicator of islet cell viability than the ADP/ATP ratio. We tested this hypothesis using mixtures of healthy and lethally heat-treated (HT) rat insulinoma cells and human islets. Measurements of ATP/DNA and ADP/ATP from the known mixtures of healthy and HT cells and islets were used to evaluate how well these parameters correlated with viability. The results indicated that ATP and ADP were rapidly (within 1 hour) depleted in HT cells. The fraction of HT cells in a mixture correlated linearly with the ATP/DNA ratio, whereas the ADP/ADP ratio was highly scattered, remaining effectively unchanged. Despite similar limitations in both ADP/ADP and ATP/DNA ratios, in that ATP levels may fluctuate significantly and reversibly with metabolic stress, the results indicated that ATP/DNA was a better measure of islet viability than the ADP/ATP ratio.

Spirometry reference values in the Brazilian population.

The aim of the present study was to provide new spirometry reference equations in a sample of the Brazilian population for the following parameters: forced vital capacity (FVC), forced expiratory volume in 1 second (FEV1), FEV1/FVC ratio, peak of expiratory flow (PEF), forced expiratory flow at 50% (FEF50%), 75% average vital capacity (FEF25-75%), and average forced expiratory flow time (FEFT). This was a prospective study using results from chest radiographs, electrocardiograms, and questionnaires to investigate the participants' respiratory symptoms, sedentarism, and comorbidities (Charlson comorbidity index). From December 2010 to July 2014, individuals were randomly selected from various locations in the state of Rio de Janeiro. All individuals were examined by a single technician in the morning at the laboratory, and performed the spirometry with the same spirometer. Spirometry values were tabulated for the creation of three equation models: linear regression, logarithmic regression, and logarithms through a method that incorporates the lambda, median, and coefficient of variation (LMS method). Initially, 7003 individuals from both genders were contacted, and 454 were recruited. The data from the new equations were compared with one Brazilian and eight international equations, resulting in a high correlation (r>0.9). The values derived from the LMS method and linear regression were very similar (P>0.5), and both could be used to acquire the reference values for Brazilian spirometry. Data derived from the equations of this study were different from the current Brazilian equation, which could be justified by the different method used.

Cross-sensitivity of methylating agents, hydroxyurea, and methotrexate in human tumor cells of the Mer- phenotype.

Five human tumor cell lines of the Mer- phenotype sensitive to killing by the methylating agent 5-(3-methyl-1-triazeno)imidazole-4-carboxamide were sensitive to hydroxyurea (HU) compared with 15 cell lines resistant to methylating agents (Mer+ phenotype). In a study using fewer cell lines, Mer- cells were also sensitive to methotrexate but not to seven other agents including the antimetabolites 1-beta-D-arabinofuranosylcytosine and 5-fluorouracil. Cells sensitive to HU were designated the Hu- phenotype. Five autologous Mer+ lines, derived in vitro by treating Mer- lines with methylating agents, did not become resistant to HU or methotrexate (Mer+ Hu- phenotype). All Mer+ lines studied had enhanced ability to reactive methylated adenovirus. Adenovirus was inactivated by prolonged treatment with HU, but no enhanced reactivation of HU-treated virus was found in Mer+ cell lines. Cell survival after 5-(3-methyl-1-triazeno)imidazole-4-carboxamide treatment was not significantly decreased by HU, nor was replication of methylated adenovirus inhibited by HU in Mer- or Mer+ lines. Replication of untreated adenovirus was poor in Mer- cells treated with HU, indicating that sensitivity of cells to HU was associated with inhibition of DNA synthesis. These results suggest that cell sensitivity to deoxynucleotide depletion is linked, perhaps coincidentally, to the Mer- phenotype. The retention of HU and methotrexate sensitivity by cells after development of resistance to 5-(3-methyl-1-triazeno)imidazole-4-carboxamide may have therapeutic implications.

Relationships among cell survival, O6-alkylguanine-DNA alkyltransferase activity, and reactivation of methylated adenovirus 5 and herpes simplex virus type 1 in human melanoma cell lines.

O6-Alkylguanine-DNA alkyltransferase (ATase) activity and host cell reactivation (HCR) of 5-(3-methyl-1-triazeno)imidazole-4-carboxamide (MTIC)-methylated viruses were compared in human melanoma cell lines that were sensitive or resistant to killing by the antitumor DNA-methylating agent MTIC. Enhanced HCR of adenovirus 5 (defined as the Mer+ phenotype) generally showed a semiquantitative correlation with the natural or induced resistance of the host cells to the toxic effects of MTIC and to the level of ATase activity. However, one MTIC-resistant cell line was found (MM170) which had a low level of ATase and intermediate HCR of adenovirus. The HCR of herpes simplex virus type 1 (HSV-1) was enhanced in the Mer+ cells that had natural resistance to MTIC compared with Mer- cells. On the other hand, HCR of HSV-1 in Mer+ cells with induced resistance to MTIC was similar to that in Mer- cells. Neither adenovirus 5 nor HSV-1 infection induced ATase activity in Mer- cells. This indicates that resistance to the toxic effects of methylating agents is not invariably associated with high levels of ATase activity in human melanoma cells. Furthermore, while induction of the Mer+ phenotype from Mer- cells was usually accompanied by the recovery of ATase activity, induced Mer+ cells had less proficient repair than natural Mer+ cells, as judged quantitatively by slightly lower cellular resistance and qualitatively by deficient HCR response for HSV-1. These results suggest that the Mer- and induced Mer+ cells lack an ATase-independent DNA repair mechanism. No differences in MTIC-induced DNA repair synthesis or strand breaks were found between the Mer-, natural Mer+, and induced Mer+ phenotypes. However, UV-induced DNA repair synthesis was higher in the natural Mer+ than in the Mer- or induced Mer+ cells, both of which had increased cellular sensitivity to the antimetabolites methotrexate and hydroxyurea. These differences may be related to the effects observed with MTIC. A wide range of ATase activities was found in human melanoma biopsy material.

Loss of promoter IV-driven BDNF expression impacts oscillatory activity during sleep, sensory information processing and fear regulation.

Posttraumatic stress disorder is characterized by hyperarousal, sensory processing impairments, sleep disturbances and altered fear regulation; phenotypes associated with changes in brain oscillatory activity. Molecules associated with activity-dependent plasticity, including brain-derived neurotrophic factor (BDNF), may regulate neural oscillations by controlling synaptic activity. BDNF synthesis includes production of multiple Bdnf transcripts, which contain distinct 5' noncoding exons. We assessed arousal, sensory processing, fear regulation and sleep in animals where BDNF expression from activity-dependent promoter IV is disrupted (Bdnf-e4 mice). Bdnf-e4 mice display sensory hyper-reactivity and impaired electrophysiological correlates of sensory information processing as measured by event-related potentials (ERP). Utilizing electroencephalogram, we identified a decrease in slow-wave activity during non-rapid eye movement sleep, suggesting impaired sleep homeostasis. Fear extinction is controlled by hippocampal-prefrontal cortical BDNF signaling, and neurophysiological communication patterns between the hippocampus (HPC) and medial prefrontal cortex (mPFC) correlate with behavioral performance during extinction. Impaired fear extinction in Bdnf-e4 mice is accompanied by increased HPC activation and decreased HPC-mPFC theta phase synchrony during early extinction, as well as increased mPFC activation during extinction recall. These results suggest that activity-dependent BDNF signaling is critical for regulating oscillatory activity, which may contribute to altered behavior.

Protection against CNS ischemia by temporary interruption of function-related processes of neurons.

Previous studies have shown that most of the energy consumption of CNS tissue is used for processes that subserve signaling functions of the cells. Since these function-related processes are probably not essential to cell viability, blocking them reversibly with a combination of pharmacologic agents should protect cells from a reduction in energy metabolism. Preliminary experiments to test this hypothesis were performed on isolated rabbit retinas. They were maintained in a newly devised chamber that permitted continuous monitoring of electrophysiological function for > or = 8 h. Ischemia was simulated by a 6-fold reduction in both O2 and glucose. This caused a rapid (t1/2 75 s) and complete loss of the light-evoked response in the optic nerve. Untreated retinas showed full recovery after 1/2 h of deprivation, but only 50% recovery after 1 h and little or no recovery after 2 or 3 h. Retinas exposed during 3 h of deprivation to a combination of six agents that abolished electrophysiologic function and reduced glucose utilization [tetrodotoxin (TTX), 2-amino-4-phosphonobutyric acid (APB), 2-amino-5-phosphonovaleric acid (APV), amiloride, Mg2+, and Li+] showed full recovery. We conclude that reducing energy requirements by blocking functional processes can prevent ischemic damage.

Importance of nitric oxide synthase inhibition to the attenuated vascular responses induced by topical L-nitroarginine during vibrissal stimulation.

We assessed the regional cerebral blood flow (rCBF) response to vibrissal stimulation before and after nitric oxide synthase (NOS) inhibitors were topically applied through a closed cranial window placed over the cortical barrel fields in anesthetized Sprague-Dawley rats. In the presence of L-nitroarginine (1 mM), both the maximum and total responses became reduced, but only in those animals demonstrating > 50% inhibition of NOS activity as determined by the conversion of [3H]arginine to [3H]citrulline within homogenates taken from cortical gray matter under the cranial window. The degree of enzyme inhibition depended in part, upon duration after topical application of NOS inhibitor. When > 50%, enzyme inhibition correlated with the decrease in maximum and total rCBF response (p < 0.01). These findings emphasize the merits of assessing enzyme activity after administering NOS inhibitors, and suggest that NO generated from parenchymal NOS activity plays an important role in the cerebrovascular response to physiologic somatosensory stimulation under the stated conditions.

Cerebral blood flow changes during cortical spreading depression are not altered by inhibition of nitric oxide synthesis.

CBF increases concomitantly with cortical spreading depression (CSD). We tested the hypothesis that CBF changes during CSD are mediated by nitric oxide (NO). Male Wistar rats (n = 23) were subjected to KCl-induced CSD before and after administration of nitric oxide synthase (NOS) inhibitors N-nitro-L-arginine (L-NNA) or N-nitro-L-arginine methyl ester (L-NAME) and in nontreated animals. CBF, CSD, and mean arterial blood pressure were recorded. Brain NOS activity was measured in vitro in control, L-NNA, and L-NAME-treated rats by the conversion of [3H]arginine to [3H]citrulline. Our data show that the NOS inhibitors did not significantly change regional CBF (rCBF) during CSD, even though cortical NOS activity was profoundly depressed and systemic arterial blood pressure was significantly increased. Our data suggest that rCBF during CSD in rats is not regulated by NO.

Prevention of nitric oxide-induced neuronal injury through the modulation of independent pathways of programmed cell death.

Neuronal injury may be dependent upon the generation of the free radical nitric oxide (NO) and the subsequent induction of programmed cell death (PCD). Although the nature of this injury may be both preventable and reversible, the underlying mechanisms that mediate PCD are not well understood. Using the agent nicotinamide as an investigative tool in primary rat hippocampal neurons, the authors examined the ability to modulate two independent components of PCD, namely the degradation of genomic DNA and the early exposure of membrane phosphatidylserine (PS) residues. Neuronal injury was determined through trypan blue dye exclusion, DNA fragmentation, externalization of membrane PS residues, cysteine protease activation, and the measurement of intracellular pH (pHi). Exposure to the NO donors SIN-1 and NOC-9 (300 micromol/L) alone rapidly increased genomic DNA fragmentation from 20 +/- 4% to 71 +/- 5% and membrane PS exposure from 14 +/- 3% to 76 +/- 9% over a 24-hour period. Administration of a neuroprotective concentration of nicotinamide (12.5 mmol/L) consistently maintained DNA integrity and prevented the progression of membrane PS exposure. Posttreatment paradigms with nicotinamide at 2, 4, and 6 hours after NO exposure further demonstrated the ability of this agent to prevent and reverse neuronal PCD. Although not dependent upon pHi, neuroprotection by nicotinamide was linked to the modulation of two independent components of neuronal PCD through the regulation of caspase 1 and caspase 3-like activities and the DNA repair enzyme poly(ADP-ribose) polymerase. The current work lays the foundation for the development of therapeutic strategies that may not only prevent the course of PCD, but may also offer the ability for the repair of neurons that have been identified through the loss of membrane asymmetry for subsequent destruction.

Possible origins and distribution of immunoreactive nitric oxide synthase-containing nerve fibers in cerebral arteries.

The distribution of perivascular nerve fibers expressing nitric oxide synthase (NOS)-immunoreactivity was examined in Sprague-Dawley and Long-Evans rats using affinity-purified rabbit antisera raised against NOS from rat cerebellum. NOS immunoreactivity was expressed within the endothelium and adventitial nerve fibers in both rat strains. Labeled axons were abundant and dense in the proximal anterior and middle cerebral arteries, but were less numerous in the caudal circle of Willis and in small pial arteries. The sphenopalatine ganglia were the major source of positive fibers in these vessels. Sectioning postganglionic parasympathetic fibers from both sphenopalatine ganglia reduced the density of NOS-immunoreactive (IR) nerve fibers by > 75% in the rostral circle of Willis. Moreover, NOS-IR was present in 70-80% of sphenopalatine ganglion cells. Twenty percent of these neurons also contained vasoactive intestinal polypeptide (VIP)-immunoreactivity. By contrast, the superior cervical ganglia did not contain NOS-IR cells. In the trigeminal ganglion, NO-IR neurons were found chiefly within the ophthalmic division; approximately 10-15% of neurons were positively labeled. Colocalization with calcitonin gene-related peptide (CGRP) was not observed. Sectioning the major trigeminal branch innervating the circle of Willis decreased positive fibers by < or = 25% in the ipsilateral vessels. In the nodose ganglion, 20-30% of neurons contained NOS-immunoreactivity, whereas less than 1% were in the C2 and C3 dorsal root ganglia. Three human circles of Willis obtained at autopsy showed sparse immunoreactive fibers, chiefly within vessels of the posterior circulation. Postmortem delay accounted for some of the reduced density. Our findings indicate that nerve fibers innervating cerebral arteries may serve as a nonendothelial source of the vasodilator nitric oxide (NO). The coexistence of NOS and VIP within sphenopalatine ganglion cells raises the possibility that two vasodilatory agents, one, a highly diffusable short-lived, low-molecular-weight molecule, and the other, a polar 28 amino acid-containing peptide, may serve as coneuromediators within the cerebral circulation.

Calcitonin gene-related peptide- and neuropeptide Y-like immunoreactivity in endothelial cells after long-term stimulation of perivascular nerves.

Since both perivascular nerves and the endothelium are involved in the control of the tone of smooth muscle in the wall of blood vessels, we decided to test whether electrical stimulation of perivascular nerves might lead to structural and immunocytochemical changes in the vascular endothelium. Long-term electrical stimulation in vivo of perivascular nerves of the rabbit central ear artery was applied. The results indicate that chronic stimulation of perivascular nerves for 10 days induced structural changes and the appearance of calcitonin gene-related peptide- and neuropeptide Y-like immunoreactivity in a subpopulation of endothelial cells.

Evoked noradrenaline release in the rabbit ear artery: enhancement by purines, attenuation by neuropeptide Y and lack of effect of calcitonin gene-related peptide.

1. Adenosine (30 microM) and its analogues 5'-N-ethylcarboxaminoadenosine (5 and 30 microM) and L-phenylisopropyladenosine (5 and 30 microM), potentiated the evoked but not spontaneous release of tritiated noradrenaline in the rabbit central ear artery. 2. Prejunctional inhibition of the evoked but not spontaneous release of tritiated noradrenaline by 100 nM neuropeptide Y is greater at 2 min than at 10 min after superfusion of the peptide. 3. Calcitonin gene-related peptide (2.63 to 263 nM) did not affect the evoked or spontaneous release of tritiated noradrenaline in this preparation. 4. These results are discussed in terms of prejunctional modulation of sympathetic transmission in the rabbit central ear artery.

Changes in purinergic responses of the rabbit isolated central ear artery after chronic electrical stimulation in vivo.

1. The effect of chronic (4-16 days) electrical stimulation (5 Hz, 0.3 ms, 4-10 V) of the great auricular nerve in vivo on sympathetic cotransmission in the rabbit isolated central ear artery was examined. 2. Chronic stimulation had no significant effect on frequency-dependent (4-60 Hz) neurogenic contractions or contractile responses induced by exogenous noradrenaline (0.1-300 microM). 3. In contrast, contractions induced by exogenous alpha, beta-methylene ATP (10.0 microM) were significantly decreased in preparations from 16-day stimulated animals in comparison with sham-operated, 4-day and 8-day chronically stimulated animal groups. 4. It is concluded that chronic electrical stimulation of nerves supplying the ear artery may lead to the selective alteration of postjunctional P2x-purinoceptor mechanisms, while the effects mediated by post-junctional alpha 1-adrenoceptors remain unchanged.

Sympathetic neurotransmission in the rabbit isolated central ear artery is affected as early as one week following a single dose of X-irradiation.

1. The short-term effect of a single dose of 4500 rad X-irradiation on sympathetic neurotransmission (involving both noradrenergic and purinergic components) was assessed in the rabbit central ear artery, 1, 4 and 6 weeks post-irradiation. 2. Neurally mediated contractions were reduced as early as 1 week post-irradiation, with responses to lower frequency stimulation being initially most affected. This suggest that the purinergic component of the contractile response is affected earlier than the adrenergic component. 3. There was no change in the amplitude or sensitivity of treated preparations to the cumulative application of noradrenaline when compared with untreated preparations. In contrast, contractions to alpha, beta-methylene ATP (1 microM), a P2-purinoceptor agonist, were significantly increased at 4 and 6 weeks post-irradiation, although not at 1 week post-irradiation. 4. There were no apparent changes in the pattern of catecholamine fluorescence as a result of irradiation. However, the tissue content of noradrenaline was significantly reduced 6 weeks post-irradiation when compared with control preparations. 5. It is concluded that damage to sympathetic cotransmission is one of the early effects of irradiation, with initial impairment predominantly of the purinergic component.

X-irradiation attenuates relaxant responses in the rabbit ear artery.

1. The relaxant actions of acetylcholine, substance P and calcitonin gene-related peptide, and the levels of neuropeptide Y and calcitonin gene-related peptide were assessed in the rabbit central ear artery 1, 4 and 6 weeks after a single dose of 45 Gy X-irradiation, a dose similar to that used clinically in intraoperative radiotherapy. 2. Relaxant responses induced by acetylcholine and substance P (both endothelium-dependent) and calcitonin gene-related peptide (endothelium-independent) were reduced, and endogenous neuropeptide Y and calcitonin gene-related peptide levels were unaffected after X-irradiation. 3. The mechanism(s) by which a single dose of 45 Gy X-irradiation may selectively damage relaxant, but not direct, contractile responses of the smooth muscle (as we have shown previously) of the rabbit central ear artery are discussed.

Gacyclidine (Beaufour-Ipsen).

Beaufour-Ipsen is developing gacyclidine (GK-11) for the potential treatment of traumatic brain injury. Phase II clinical trials of the compound for this indication had been completed as of October 1999 and the company is looking for a partnership before undertaking further clinical development for this and, possibly, other indications [344879], [346265], [386763]. Phase II trials for acute spinal cord injury gave disappointing results and development for this indication has been discontinued [344879].

Effects of thermal endometrial ablation. Clinicopathologic correlations.

Electrothermal ablation of endometrium was performed on 207 women for abnormal and sometimes painful uterine bleeding. Recurrence of symptoms or inadequacy of treatment led to hysterectomy in 33 women at intervals of 2 weeks to 60 months. Patterns of uterine tissue destruction, healing and repair, and endometrial regeneration were correlated with the postablation interval. The endometrial thickness was completely destroyed. Necrosis dominated the first month, and a chronic repair-regeneration phase followed. Foreign body giant cells reacted with necrotic eschar, remnants of which were present to 47 months. Intact endometrium regenerated focally in 10 uteri and diffusely in 5. Indications for hysterectomy were attributed directly to ablation failure in 18 of 33 cases. Familiarity with the morphologic effects of ablation will aid in relating ablation failures to direct mechanisms, complications of the procedure, or unrelated events.

Delayed multidose treatment with nicotinamide extends the degree and duration of neuroprotection by reducing infarction and improving behavioral scores up to two weeks following transient focal cerebral ischemia in Wistar rats.

A single, delayed dose of nicotinamide (NAm) was shown to be protective against focal cerebral ischemia in rats, but the protection was limited to three to seven days following stroke. The investigation reported here was conducted to examine if the use of multiple doses of NAm, administered after the onset of focal cerebral ischemia, would extend the duration of neuroprotection compared with a single dose treatment regimen. Male Wistar rats were subjected to transient focal cerebral ischemia by occluding the right middle cerebral artery (MCAo) for two hours. Following MCAo, motor and sensory behavioral tests were performed daily and the cerebral infarct volumes were measured at two weeks after sacrifice. Each animal was placed into one of four groups that received either normal saline alone (Group S), one (Group A), two (Group B), or three (Group C) doses of NAm (500 mg/kg). Each animal, therefore, received three treatments over two weeks, with the first dose administered intravenously two hours after the onset of MCAo. Single and multiple doses of NAm reduced the infarction (p < 0.01) and improved (p < 0.05) the neurologic sensory and motor behavior when compared with the saline-treated animals up to two weeks after stroke. Moreover, animals that received multiple doses of NAm recuperated full motor function not different from normal, preoperative motor behavior. Delayed treatment with NAm given as multiple doses, therefore, further enhances the extent and duration of neuroprotection by significantly reducing cerebral infarct volumes, improving neurologic behavioral scores, and confers a complete motor recovery up to two weeks from the onset of focal cerebral ischemia in Wistar rats.

Neuroprotection against ischemia by metabolic inhibition revisited. A comparison of hypothermia, a pharmacologic cocktail and magnesium plus mexiletine.

Previous studies have suggested that metabolic inhibition is neuroprotective, but little evidence has been provided to support this proposal. Using the in vitro rabbit retina preparation as an established model of the central nervous system (CNS), we measured the rate of glucose utilization and lactate production, and the light-evoked compound action potentials (CAPs) as indices of neuronal energy metabolism and electrophysiologic function, respectively. We examined the effect of three (3) treatments options: hypothermia (i.e., 33 degrees C and 30 degrees C), a six-member pharmacologic "cocktail" (tetrodotoxin (0.1 microM), 2-amino-4-phosphonobutyric acid (20 microM), 2-amino-5-phosphonovaleric acid (1 mM), amiloride (1 mM), magnesium (10 mM) and lithium (10 mM) and the combination of magnesium (Mg2+ 1 mM) and mexiletine (Mex, 300 microM) on in vitro rabbit retinas, to see if there is a correlation between neuronal energy metabolism during ischemia (simulated by the reduction of oxygen from 95% to 15% and glucose from 6 mM to 1 mM), and the subsequent recovery of function. Hypothermia and the "cocktail" significantly inhibited both the rate of glucose utilization and lactate production, whereas Mg2+ and/or Mex showed only a nonsignificant tendency toward a reduction, compared to control retinas. Recovery of light-evoked CAPs was significantly improved in hypothermia- and cocktail-treated retinas, as well as with retinas exposed to the combination of Mg2+ plus Mex, but not with Mg2+ or Mex alone, relative to control retinas. A linear regression analysis of the % recovery of function versus the % reduction in the rate of glucose utilization during ischemia showed a significant correlation (r2 = 0.80, correlation coefficient = 0.9, p < 0.05) between these two parameters. This and other data discussed provide convincing evidence that there is a correlation between metabolic inhibition, achieved during ischemia, and neuroprotection.