Brain glucocorticoid receptors are necessary for the rhythmic expression of the clock protein, PERIOD2, in the central extended amygdala in mice.

The adrenal glucocorticoid, corticosterone, induces changes in gene expression in both neural and non-neural tissues. The rhythmic release of corticosterone has been shown in rats to be necessary for the rhythmic expression of the clock protein PERIOD2 (PER2) in select regions of the limbic forebrain. The mechanisms mediating the effects of glucocorticoids on changes in gene expression have been linked to the transcriptional activity of the low affinity glucocorticoid receptor, GR. We examined the patterns of PER2 expression in the brains of mice containing an inactivation of GR gene restricted to neural tissues (GR(NesCre) mice). We found that central deletion of the GR gene blunts the daily pattern of PER2 expression in the oval nucleus of the bed nucleus of the stria terminalis (BNSTov) and central nucleus of the amygdala (CEA) both of which make up the central extended amygdala, but not in the suprachiasmatic nucleus (SCN), basolateral amygdala (BLA) or dentate gyrus of the hippocampus (DG). These results implicate brain GR receptors in the regulation of PER2 expression in the BNSTov and CEA and are consistent with our previous findings that the rhythmic expression of PER2 in these areas is selectively sensitive to fluctuations in circulating corticosterone.

Timed restricted feeding restores the rhythms of expression of the clock protein, Period2, in the oval nucleus of the bed nucleus of the stria terminalis and central nucleus of the amygdala in adrenalectomized rats.

Feeding schedules that limit food availability to a set time of day are powerful synchronizers of the rhythms of expression of the circadian clock protein Period 2 (PER2) in the limbic forebrain in rats. Little is known, however, about the mechanisms that mediate the effect of such timed restricted feeding (TRF) schedules on the expression of PER2. Adrenal glucocorticoids have been implicated in the circadian regulation of clock genes expression in peripheral tissues as well as in the control of the rhythms of expression of PER2 in certain limbic forebrain regions, such as the oval nucleus of the bed nucleus of the stria terminalis (BNSTov) and central nucleus of the amygdala (CEA) in rats. To study the possible involvement of glucocorticoids in the regulation of PER2 expression by TRF, we assessed the effect of adrenalectomy on TRF-entrained PER2 rhythms in the limbic forebrain in rats. Adrenalectomy selectively abolished the rhythms of PER2 in the BNSTov and CEA in normally fed rats, as previously shown, but had no effect on TRF-entrained PER2 rhythms in the same structures. These findings show that the effect of TRF on PER2 rhythms in the limbic forebrain is independent of adrenal glucocorticoids and demonstrate that the involvement of glucocorticoids in the regulation PER2 rhythms in the limbic forebrain is not only region specific, as previously shown, but also state dependent.

[Nutritional status evaluation in maintenance hemodialysis patients]. / Evaluarea starii de nutritie la pacientii cu insuficienta renala cronica terminala tratati prin hemodializa.

UNLABELLED: Protein-calorie malnutrition is a common complication and an important predictive factor for mortality in patients with end-stage renal disease on maintenance dialysis. Therefore, nutritional status needs to be regularly assessed in these patients by using several methods. If malnutrition is diagnosed, its causes should be thoroughly searched for and properly treated. MATERIAL AND METHOD: This cross-section study aimed at evaluating the nutritional status and the possible risk factors for malnutrition in 149 (82 men) hemodialysis patients by anthropometry, biochemical tests and bioelectrical impedance analysis (BIA). The patients' height (H), post-dialysis body weight (BW), mid-arm circumference (MAC), tricipital skin-fold thickness (TST) were measured and a 3-category subjective global assessment (SGA) was performed. Body mass index (BMI), mid-arm muscle circumference (MAMC), corrected mid-arm muscle area (cMAMA) and anthropometry-estimated percent body muscle mass (% AMM) were calculated from the above measurements by using specific equations. Biochemical tests included protein equivalent of nitrogen appearance (nPNA), and predialysis serum albumin, creatinine, total cholesterol, bicarbonate, and hemoglobin (Hb) levels. We used BIA to estimate body composition - i.e. percent body fat (% BBF), fat-free mass (% FFM), body cell mass (% BCM), extracellular mass (% ECM), muscle mass (% BMM)--and the phase angle (PhA). T-test was used to make comparisons and Pearson coefficient to analyze the correlations. P < 0.05 was considered statistically significant. RESULTS: The male patients had a higher mean muscle mass--as estimated by serum creatinine (9.8 s 8.3 mg/dl; P < 0.0001) and by % BMM (41.7% vs 34.7%)--and a lower fat mass--as estimated by TST (0.95 cm vs 1.2 cm; P = 0.016) and by % FAT (16.7% vs 31.3%; P < 0.0001) than the female patients. Age was found to be positively correlated with BMI (P = 0.001), but inversely correlated with % BCM (P = 0.013) and with % AMM (P = 0.003). Patients with diabetes had lower % BCM than those without diabetes (32.9 vs 35.9%; P = 0.041). The presence of heart failure was associated with significantly reduced MAMC (22.0 vs 23.6 cm2; P = 0.045), % AMM (28.5 vs 32.1; P = 0.021), % BCM (33.0 vs 36.1% ; P = 0.034), BMM/H2 (8.6 vs 9.4 kg/m2; P = 0.013), nPNA (1.17 vs 1.34 g/kg-d ; P = 0.047), serum albumin (39.7 vs 42.4 g/l; P = 0.010), serum creatinine (8.1 vs 9.4 mg/dl; P = 0.008) and Hb (10.5 vs 11.2 g/dl; P = 0.017). The serum Hb level was positively correlated with BMI (P = 0.005), BMM/H2 (P = 0.009), serum albumin (P = 0.002) and serum creatinine (P = 0.011). Also, patients with category B-SGA were older (63.7 vs 50.1 y.o.; P < 0.0001) and had more heart failure (42% vs 13%; P = 0.013) than those with category A-SGA. In hemodialysis patients, advancing age, diabetes, heart failure and decreasing Hb levels are associated with worse nutritional status, as estimated by anthropometry, biochemical markers and BIA. Whether treatment of comorbidities such as heart disease and anemia may improve nutritional status in these patients is an important issue that deserves further research.

Glucocorticoid rhythms control the rhythm of expression of the clock protein, Period2, in oval nucleus of the bed nucleus of the stria terminalis and central nucleus of the amygdala in rats.

We investigated the involvement of the adrenal glucocorticoid, corticosterone, in the control of the rhythmic expression of the circadian clock protein, Period2, in forebrain nuclei known to be sensitive to glucocorticoids, stressors and drugs of abuse, the oval nucleus of the bed nucleus of the stria terminalis and the central nucleus of the amygdala. We found previously that the daily rhythm of Period2 in these nuclei is uniquely dependent on the integrity of the adrenal glands (Amir S, Lamont EW, Robinson B, Stewart J (2004) A circadian rhythm in the expression of PERIOD2 protein reveals a novel SCN-controlled oscillator in the oval nucleus of the bed nucleus of the stria terminalis. J Neurosci 24:781-790; Lamont EW, Robinson B, Stewart J, Amir S (2005) The central and basolateral nuclei of the amygdala exhibit opposite diurnal rhythms of expression of the clock protein Period2. Proc Natl Acad Sci U S A 102:4180-4184). We now show that, in rats, in the absence of the adrenals, corticosterone replacement via the drinking water, which is associated with daily fluctuations in corticosterone levels, restores the rhythm of Period2 in the oval nucleus of the bed nucleus of the stria terminalis and central nucleus of the amygdala. Corticosterone replacement via constant-release pellets has no effect. These results underscore the importance of circadian glucocorticoid signaling in Period2 rhythms in the oval nucleus of the bed nucleus of the stria terminalis and central nucleus of the amygdala and suggest a novel mechanism whereby stressors, drugs of abuse, and other abnormal states that affect the patterns of circulating glucocorticoids can alter the functional output of these nuclei.

[The first case of coronary artery by-pass grafting surgery (CABG) in a patient on maintenance dialysis in Iasi]. / Prima operatie de by-pass coronarian la un pacient dializat cronic în Iasi.

Coronary artery disease has a significantly higher prevalence in chronic dialysis patients compared to the general population, explained by a cluster of non-specific and specific (uremia-associated) cardiovascular risk factors, typical for these patients. Nephrologists and cardiovascular surgeons worldwide are rather reluctant to offer CABG to dialysis patients, because of concerns about higher risks associated with this procedure in this frail population. However, there is an increasing opinion supporting a more aggressive management of coronary artery disease in uremic individuals. To illustrate this "positive attitude", we report here the first dialysis patient ever treated by CABG in Iasi; his good outcome was both rewarding and encouraging for us all.

Transbronchial administration of adenoviral-mediated interleukin-10 gene to the donor improves function in a pig lung transplant model.

Interleukin-10 (IL-10) gene transfection of donor lungs prior to transplantation is an attractive strategy to reduce ischemia-reperfusion induced lung injury. However, experimental data with gene therapy in large animal models of lung transplantation are generally lacking. We have developed a simple clinically applicable technique for adenoviral-mediated gene delivery of human IL-10 to the lung of large animals that provides homogenous gene expression after 12-24 h of transfection. Using this technique of gene delivery, we have studied the dynamics of adenoviral gene delivery to the lung in the setting of lung transplantation. Although there is a persistent inflammatory response to the adenoviral vector, we achieved significant expression of human IL-10 in lung tissue before lung retrieval to obviate the deleterious impact of the adenoviral vector on the donor lung. The administration of adenoviral-mediated human IL-10 to the donor lung reduced ischemia-reperfusion injury and improved graft function after lung transplantation in this pig lung transplantation model. Transfection of adenoviral-mediated human IL-10 to the donor lung prevented the release of inflammatory cytokines such as IL-6 in lung tissue and plasma. We have demonstrated that IL-10 gene therapy has significant potential to prevent or treat the inflammatory response associated with ischemia-reperfusion injury in lung transplantation. In the future, IL-10 gene therapy could also be used for immunomodulation or tolerance induction.

A clinical description of rifampicin-induced acute renal failure in 170 consecutive cases.

Rifampicin re-administration may cause immunologically mediated acute tubulo-interstitial injury. Retrospectively, 170 consecutive cases with acute renal failure (ARF) following re-treatment with rifampicin (71% males, 29% females, age 21 to 68 years) were analysed, which accounted for 12% of all ARF patients treated by two large dialysis referral centres in Romania, Timisoara and Iasi, between 1974-2001 and 1988-2001, respectively. The most frequent clinical features of rifampicin-induced ARF were: Anuria, gastro-intestinal (abdominal pain, nausea, vomiting and diarrhoea) and "flu-like" symptoms. Urine analysis revealed sterile leucocyturia in 54%, proteinuria in 31%, haematuria in 26% and haemoglobinuria in 7% of cases. Haemolytic anaemia was frequent, found in 66% of the patients; half of these had Hct values of < 30%, thrombocytopenia and also more severe renal damage (a longer anuric phase and a slower recovery of the renal function), thus suggesting a severe multi-target autoimmune aggression. The association of hepatic injury--not explained by prior hepatic disease, B or C hepatitis virus infection or history of alcohol abuse--was encountered in 17% of the cases, without a significant influence on the renal and the general outcome. The outcome of rifampicin-induced ARF is generally favourable, with complete recovery of the renal function within 30 days in 52% of the cases and within 90 days in 92% of the cases. The mortality rate was 3.5%, compared to 21% for the overall ARF population treated during the same period (p < 0.05).

Mechanistic basis for kinetic differences between the rat alpha 1, alpha 2, and alpha 3 isoforms of the Na,K-ATPase.

Previous studies showed that the alpha 1, alpha 2, and alpha 3 isoforms of the catalytic subunit of the Na,K-ATPase differ in their apparent affinities for the ligands ATP, Na(+), and K(+). For the rat isoforms transfected into HeLa cells, K'(ATP) for ATP binding at its low affinity site is lower for alpha 2 and alpha 3 compared with alpha 1; relative to alpha 1 and alpha 2, alpha 3 has a higher K'(Na) and lower K'(K) (Jewell, E. A., and Lingrel, J. B (1991) J. Biol. Chem. 266, 16925--16930; Munzer, J. S., Daly, S. E., Jewell-Motz, E. A., Lingrel, J. B, and Blostein, R. (1994) J. Biol. Chem. 269, 16668--16676). The experiments described in the present study provide insight into the mechanistic basis for these differences. The results show that alpha 2 differs from alpha1 primarily by a shift in the E(1) E(2) equilibrium in favor of E(1) form(s) as evidenced by (i) a approximately 20-fold increase in IC(50) for vanadate, (ii) decreased catalytic turnover, and (iii) notable stability of Na,K-ATPase activity at acidic pH. In contrast, despite its lower K'(ATP) compared with alpha 1, the E(1) E(2) poise of alpha 3 is not shifted toward E(1). Distinct intrinsic interactions with Na(+) ions are underscored by the marked selectivity for Na(+) over Li(+) of alpha 3 compared with either alpha1 or alpha 2 and higher K'(Na) for cytoplasmic Na(+), which persists over a 100-fold range in proton concentration, independent of the presence of K(+). The kinetic analysis also suggests alpha 3-specific differences in relative rates of partial reactions, which impact this isoform's distinct apparent affinities for both Na(+) and K(+).

Glucose down-regulates the expression of the peroxisome proliferator-activated receptor-alpha gene in the pancreatic beta -cell.

To better understand the action of glucose on fatty acid metabolism in the beta-cell and the link between chronically elevated glucose or fatty acids and beta-cell decompensation in adipogenic diabetes, we investigated whether glucose regulates peroxisomal proliferator-activated receptor (PPAR) gene expression in the beta-cell. Islets or INS(832/13) beta-cells exposed to high glucose show a 60-80% reduction in PPARalpha mRNA expression. Oleate, either in the absence or presence of glucose, has no effect. The action of glucose is dose-dependent in the 6-20 mm range and maximal after 6 h. Glucose also causes quantitatively similar reductions in PPARalpha protein and DNA binding activity of this transcription factor. The effect of glucose is blocked by the glucokinase inhibitor mannoheptulose, is partially mimicked by 2-deoxyglucose, and is not blocked by the 3-O-methyl or the 6-deoxy analogues of the sugar that are not phosphorylated. Chronic elevated glucose reduces the expression levels of the PPAR target genes, uncoupling protein 2 and acyl-CoA oxidase, which are involved in fat oxidation and lipid detoxification. A 3-day exposure of INS-1 cells to elevated glucose results in a permanent rise in malonyl-CoA, the inhibition of fat oxidation, and the promotion of fatty acid esterification processes and causes elevated insulin secretion at low glucose. The results suggest that a reduction in PPARalpha gene expression together with a rise in malonyl-CoA plays a role in the coordinated adaptation of beta-cell glucose and lipid metabolism to hyperglycemia and may be implicated in the mechanism of beta-cell "glucolipotoxicity."

Lipid rather than glucose metabolism is implicated in altered insulin secretion caused by oleate in INS-1 cells.

A comprehensive metabolic study was carried out to understand how chronic exposure of pancreatic beta-cells to fatty acids causes high basal secretion and impairs glucose-induced insulin release. INS-1 beta-cells were exposed to 0.4 mM oleate for 3 days and subsequently incubated at 5 or 25 mM glucose, after which various parameters were measured. Chronic oleate promoted triglyceride deposition, increased fatty acid oxidation and esterification, and reduced malonyl-CoA at low glucose in association with elevated basal O(2) consumption and redox state. Oleate caused a modest (25%) reduction in glucose oxidation but did not affect glucose usage, the glucose 6-phosphate and citrate contents, and the activity of pyruvate dehydrogenase of INS-1 cells. Thus changes in glucose metabolism and a Randle-glucose/fatty acid cycle do not explain the altered secretory properties of beta-cells exposed to fatty acids. The main response of INS-1 cells to chronic oleate, which is to increase the oxidation and esterification of fatty acids, may contribute to cause high basal insulin secretion via increased production of reducing equivalents and/or the generation of complex lipid messenger molecule(s).

Molecular or pharmacologic perturbation of the link between glucose and lipid metabolism is without effect on glucose-stimulated insulin secretion. A re-evaluation of the long-chain acyl-CoA hypothesis.

The mechanism by which glucose stimulates insulin secretion from the pancreatic islets of Langerhans is incompletely understood. It has been suggested that malonyl-CoA plays a regulatory role by inhibiting fatty acid oxidation and promoting accumulation of cytosolic long-chain acyl-CoA (LC-CoA). In the current study, we have re-evaluated this "long-chain acyl-CoA hypothesis" by using molecular and pharmacologic methods to perturb lipid metabolism in INS-1 insulinoma cells or rat islets during glucose stimulation. First, we constructed a recombinant adenovirus containing the cDNA encoding malonyl-CoA decarboxylase (AdCMV-MCD), an enzyme that decarboxylates malonyl-CoA to acetyl-CoA. INS-1 cells treated with AdCMV-MCD had dramatically lowered intracellular malonyl CoA levels compared with AdCMV-betaGal-treated cells at both 3 and 20 mM glucose. Further, at 20 mM glucose, AdCMV-MCD-treated cells were less effective at suppressing [1-14C]palmitate oxidation and incorporated 43% less labeled palmitate and 50% less labeled glucose into cellular lipids than either AdCMV-betaGAL-treated or untreated INS-1 cells. Despite the large metabolic changes caused by expression of MCD, insulin secretion in response to glucose was unaltered relative to controls. The alternative, pharmacologic approach for perturbing lipid metabolism was to use triacsin C to inhibit long-chain acyl-CoA synthetase. This agent caused potent attenuation of palmitate oxidation and glucose or palmitate incorporation into cellular lipids and also caused a 47% decrease in total LC-CoA. Despite this, the drug had no effect on glucose-stimulated insulin secretion in islets or INS-1 cells. We conclude that significant disruption of the link between glucose and lipid metabolism does not impair glucose-stimulated insulin secretion in pancreatic islets or INS-1 cells.

Rifampicin-induced acute renal failure: a series of 60 patients.

BACKGROUND: Since 1971, 55 case-reports of rifampicin-induced ARF have been published, but systematic data on this condition are not available, in view of the disparate nature of the observations. METHODS: We retrospectively assessed prevalence, clinical and biochemical features, and prognostic factors of 60 consecutive cases (41 males/19 females, age 22-68 years), who were admitted to the Iasi Dialysis Centre from 1987 to 1995 for acute renal failure (ARF) following re-treatment with rifampicin. RESULTS: The clinical appearance consisted mainly of gastrointestinal and 'flu-like' symptoms and clinical signs of intravascular haemolysis (the latter in 17% of cases). Frequent laboratory findings were anaemia (96% of cases), leukocytosis (63%), and thrombocytopenia (50%). Severe anaemia was associated with marked haemolysis (25% cases), thrombocytopenia, longer anuria, and slower rate of renal function recovery. Signs of hepatic injury were found in 25% of patients, but it did not seem to affect the outcome of renal function. Prognostic factors in post-rifampicin ARF proved to be the following: the duration of the anuric phase (correlated with the number of dialysis sessions and with the rate of decrease of azotaemia) and the severity of the immunological abnormalities and inflammatory syndrome (haemolysis, leukocytosis, hypergammaglobulinaemia). Post-rifampicin ARF accounted for 16.6% of all ARF cases hospitalized in our Centre during the studied period. Its clinical course was favourable; the mortality rate was only 1.6% (1 case), compared to a 20% general mortality rate among all ARF patients. Full recovery of renal function was achieved in 40% and 96% of patients, 30 and 90 days respectively from onset. CONCLUSIONS: ARF after treatment with rifampicin is not an uncommon condition, especially when tuberculosis prevalence is high, but renal prognosis is usually favourable. Thrombocytopenia, immune haemolytic anaemia, and intravascular haemolysis are frequent complications which are associated with a more severe renal injury.

Deglutition in elderly patients with dementia: findings of videofluorographic evaluation and impact on staging and management.

Oral and pharyngeal function in 131 institutionalized elderly patients with advanced dementia was evaluated by means of videofluoroscopic deglutition examination (VDE). Findings were normal in only nine (7%) patients. Oral-stage dysfunction was observed in 93 (71%) patients, pharyngeal dysfunction in 56 (43%), and pharyngoesophageal-segment abnormalities in 43 (33%). Multiple-stage dysfunction was noted in 55 (42%) patients. Major aspiration of contrast medium was present in 31 patients, and minor aspiration in 66. Evaluation of VDE findings prompted a change in clinical staging (degree of impairment) in 40 patients and substantial alteration in treatment planning in 28. At clinical bedside evaluation, the degree of bolus misdirection was overestimated in 19 patients with minor aspiration and underestimated in seven with major aspiration. Dementia is often associated with oral and pharyngeal impairment, and VDE can be important in diagnosis and treatment.

Aspiration and the elderly.

Aspiration is prevalent in the elderly but its association with impairment of oral intake and gastroesophageal reflux is often misunderstood. This paper describes the causes, pathophysiology, and consequences of aspiration and their unique features in aged persons. It also explains how videofluoroscopic evaluation can assess current function while limiting factors that result in misinformation. The management of aspiration is discussed, emphasizing the importance and difficulties in maintaining functional well-being and possible complications of therapy.

Comparison of two methods of observing work areas in a hospital pharmacy.

Two methods of observing the work area within a pharmacy of an acute care, 140-bed hospital were compared. A work activity recording form was developed from the literature and from discussion with the pharmacy staff. The instrument was coded, tested, and refined in a pilot study. One hospital pharmacy department work area was personally observed for a period of time equal to an entire work week. A time-motion camera was placed in the pharmacy which recorded the activities in the same pharmacy work area for a second work week. The results indicated that the two methods of observation were approximately equivalent for observing the work activities of the nonpharmacists but not for the work activities of the pharmacists. The camera method of observing work areas of a hospital pharmacy appeared to be reliable and inexpensive, but it lacked the precision obtained with the personal observation mode. Further use of time-motion cameras within the hospital pharmacy setting are indicated.