Stable expression of type I gamma-aminobutyric acidA/benzodiazepine receptors in a transfected cell line.

Expression plasmids were constructed with cDNAs encoding the rat gamma-aminobutyric acid-A (GABAA) receptor alpha 1, beta 2, and gamma 2 subunits and were cotransfected into cultured human embryonic kidney 293 cells. A single cell line (WS-1) was established after G-418 treatment and clonal selection. This cell line contained saturable, high affinity binding sites for the benzodiazepines [3H] Ro 15-4513 and [3H]flunitrazepam that were modulated by GABA. Competition experiments with benzodiazepine receptor ligands suggest a profile characteristic of native "type I" benzodiazepine receptors, because strong correlations were observed between the Ki values of these ligands in WS-1 cells and in both cerebellar homogenates (r = 0.97, p < 0.0001) and 293 cells transiently transfected with the corresponding cDNAs (r = 0.96, p < 0.001). Fluorescence intensity in WS-1 cells loaded with the Cl(-)-specific probe 6-methoxy-N-(3-sulfopropyl)-quinolinium was reliably increased by GABA. This effect was blocked by bicuculline and augmented by midazolam, consistent with the presence of GABA-gated, benzodiazepine receptor-modulated, Cl- channels. Northern blot analysis revealed the presence of mRNAs encoding alpha 1 and gamma 2 receptor subunits. Southern blot analysis confirmed genomic integration of transfected alpha 1 and gamma 2 cDNAs. The beta 2 subunit was not detected in either Northern or Southern blot analysis, indicating that a functional type I GABAA/benzodiazepine receptor complex can be constituted without a beta subunit.     

Cerebrospinal fluid viral load is related to cortical atrophy and not to intracerebral calcifications in children with symptomatic HIV disease

The relationships between viral load in plasma and cerebrospinal fluid (CSF) and computed tomography (CT) brain scan abnormalities were studied in 39 children between 0.5 and 13 years of age with symptomatic HIV-1 disease. Quantitative RNA PCR was used to determine HIV-1 RNA levels and a semiquantitative analog rating technique was used to evaluate non-contrast CT brain scans. CSF HIV-1 RNA copy number correlated significantly with CT brain scan ratings for severity of cortical atrophy (r = 0.36; P < 0.05) but not with ratings of intracerebral calcifications (r = -12; NS). The difference between these two correlations was significant (P < 0.05). Plasma HIV-1 RNA copy number did not correlate significantly with any CT brain scan ratings or with CSF viral load (r = 0.05; NS). Severity of cortical atrophy appeared to reflect the level of viral load in the CSF, supporting the notion that active HIV-1 replication in the CNS is at least in part responsible for such brain abnormalities in children. The lack of correlation of intracerebral calcifications with other CT brain scan abnormalities as well as with CSF viral load suggests that this lesion is relatively independent and may reflect a different neuropathologic process.     

Tumor lysis syndrome in widely metastatic small-cell lung cancer

Tumor lysis syndrome is a rare complication of nonhematologic malignancies that results from massive necrosis of neoplastic cells after chemotherapy. This syndrome consists of life-threatening metabolic derangements, including severe hyperphosphatemia, hyperkalemia, hyperuricemia, and hypocalcemia, and may result in renal failure and death if not recognized early and treated appropriately. We report a case of tumor lysis syndrome after induction chemotherapy in a patient with widely metastatic smallcell lung cancer. This case emphasizes the importance of awareness and early treatment of this syndrome.     

Mutations in the tyrosine kinase domain of the epidermal growth factor receptor in non-small cell lung cancer.

We evaluated somatic genetic alterations in the kinase domain of the EGFR gene in the tumors of 219 non-small cell lung cancer patients of primarily Caucasian and African American origins. We identified 26 patients (12%) whose tumors had a mutation in the EGFR gene, and 11 (5%) patients carried novel genomic variations consistent with germ-line polymorphisms. All but one mutation were identified in Caucasian patients affected with adenocarcinoma. EGFR mutations were more frequent in women and in nonsmokers, but a significant portion of the affected patients were men (12 of 26) and current or past smokers accounted for half of the patients affected (13 of 26). Screening subjects with EGFR mutations may identify patients whose tumors could respond to targeted therapy using tyrosine kinase inhibitors.     

Methylglyoxal chronic administration promotes diabetes-like cardiac ischaemia disease in Wistar normal rats

Background and aimsThe influence of lifestyle is well documented, especially the diet regime, in the development of type 2 diabetes (T2D) and associated cardiovascular diseases. Diabetic patients have increased risk of suffering cardiac ischemia and impaired response to such accidents. Methylglyoxal (MG) circulates at high concentration in diabetics' blood and is linked to the development of diabetes chronic complications. We propose that besides promoting the cardiovascular disease, MG may also negatively regulate the endogenous cardioprotection pathways after ischemia.Methods and resultsWe performed a comparative study between three animal groups: normal Wistar (W), type 2 diabetic non-obese Goto-Kakizaki (GK) and normal rats submitted to MG chronic administration (3months) with gradually enhanced concentration, up to 75 mg/Kg (WMG). Hearts were submitted to different experimental conditions: control, ischemia and ischemia-reperfusion. Levels of oxidative stress markers, advanced glycation end-products (AGEs) and their receptors (RAGEs) were evaluated. The serine/threonine protein kinase Akt (Akt), crucial for cardiomyocytes recovery after ischemia, and apoptosis markers were also assessed.Levels of MG, systemic and cardiac oxidative stress markers, AGEs and RAGEs were similar in GK and WMG groups. Akt protein was negatively regulated by MG, leading to impaired apoptotic markers.ConclusionChronic MG administration to normal rodents mimicked most diabetic alterations, being associated with the development of cardiovascular disease and the impairment of survival pathways. Our results demonstrate the negative effect of MG rich diet in healthy animals and suggest the potential of methylglyoxal as a therapeutic target in diabetes.     

First Japanese case of atypical progeroid syndrome/atypical Werner syndrome with heterozygous LMNA mutation

Atypical progeroid syndrome (APS), including atypical Werner syndrome (AWS), is a progeroid syndrome involving heterozygous mutations in the LMNA gene encoding the nuclear protein lamin A/C. We report the first Japanese case of APS/AWS with a LMNA mutation (p.D300N). A 53‐year‐old Japanese man had a history of recurrent severe cardiovascular diseases as well as brain infarction and hemorrhages. Although our APS/AWS patient had overlapping features with Werner syndrome (WS), such as high‐pitched voice, scleroderma, lipoatrophy and atherosclerosis, several cardinal features of WS, including short stature, premature graying/alopecia, cataract, bird‐like face, flat feet, hyperkeratosis on the soles and diabetes mellitus, were absent. In immunofluorescence staining and electron microscopic analyses of the patient's cultured fibroblasts, abnormal nuclear morphology, an increase in small aggregation of heterochromatin and a decrease in interchromatin granules in nuclei of fibroblasts were observed, suggesting that abnormal nuclear morphology and chromatin disorganization may be associated with the pathogenesis of APS/AWS.     

Melatonin improves insulin resistance and hepatic steatosis through attenuation of alpha‐2‐HS‐glycoprotein

Melatonin plays an important role in regulating circadian rhythms. It also acts as a potent antioxidant and regulates glucose and lipid metabolism, although the exact action mechanism is not clear. The α2‐HS‐glycoprotein gene (AHSG) and its protein, fetuin‐A (FETUA), are one of the hepatokines and are known to be associated with insulin resistance and type 2 diabetes. The aim of this study was to determine whether melatonin improves hepatic insulin resistance and hepatic steatosis in a FETUA‐dependent manner. In HepG2 cells treated with 300 μmol/L of palmitic acid, phosphorylated AKT expression decreased, and FETUA expression increased, but this effect was inhibited by treatment with 10 μmol/L of melatonin. However, melatonin did not improve insulin resistance in FETUA‐overexpressing cells, indicating that improvement in insulin resistance by melatonin was dependent on downregulation of FETUA. Moreover, melatonin decreased palmitic acid‐induced ER stress markers, CHOP, Bip, ATF‐6, XBP‐1, ATF‐4, and PERK. In addition, in the high‐fat diet (HFD) mice, oral treatment with 100 mg/kg/day melatonin for 10 weeks reduced body weight gain to one‐third of that of the HFD group and hepatic steatosis. Insulin sensitivity and glucose intolerance improved with the upregulation of muscle p‐AKT protein expression. FETUA expression and ER stress markers in the liver and serum of HFD mice were decreased by melatonin treatment. In conclusion, melatonin can improve hepatic insulin resistance and hepatic steatosis through reduction in ER stress and the resultant AHSG expression.