Establishment of FSH-responsive cell lines by transfection of pre-ovulatory human granulosa cells with mutated p53 (p53val135) and Ha-ras genes.

Human granulosa cells were immortalized by transfection of the primary cells with a mutated p53 gene in combination with the Harvey-ras oncogene, yielding established cell lines designated HGP53. Here we report that forskolin, 8-Br-cAMP and FSH modulate cell growth and steroidogenesis in HGP53 cells. Low concentrations of 8-Br-cAMP or FSH stimulated cell proliferation, while higher doses attenuated cell proliferation. Progesterone production was already evident at an FSH concentration of 0.3 mIU/ml and was maximally stimulated (50-135-fold) at 50 mIU/ml of FSH. Expression levels of steroidogenic acute regulatory protein (StAR), adrenodoxin and cytochrome P450scc were enhanced 64-, 48- and 3.1-fold respectively by FSH stimulation. Dexamethasone enhanced FSH/cAMP-induced steroidogenesis and this effect involved a marked elevation in the intracellular level of adrenodoxin and P450scc, concomitantly with a marked decrease in StAR. Conversely, basic fibroblast growth factor attenuated FSH-stimulated progesterone production, and this effect involved reductions in adrenodoxin, P450scc and StAR levels. These data suggest that the rate of steroidogenesis may be determined by the ratio of StAR and P450scc, rather than by the level of each protein alone. Whereas FSH at a low dose slightly reduced apoptosis induced by serum withdrawal from HGP53 cells, higher doses enhanced it. Dexamethasone dramatically attenuated FSH- or forskolin-enhanced apoptosis. In conclusion, FSH-dependent mechanisms of differentiation, luteinization and apoptosis can be preserved in human granulosa cells immortalized by mutated p53. Moreover, this system lends itself to studies on cross-talk between the endocrine and paracrine factors that control these processes.     

Further characterization of the slow muscarinic responses inXenopus oocytes

In immature follicular ocytes of the frogXenopus laevis, application of muscarinic agonists evokes a complex response consisting of a fast and a slow Cl currents (the dominant responses), Cl current fluctuations, and a less prominent slow K current. The characteristics of the slow ACh-evoked potassium current were studied using the two-electrode voltage clamp method, and compared to those of the ACh-evoked Cl currents. In experiments designed to study the K current response separately, without the interference of ACh-evoked Cl currents, the holding potential was set close or equal to Cl equilibrium potential (measured as the reversal potential of the ACh-evoked Cl current). The Cl current responses were studied in cells that had negligible K current response. The dose-response curve of the potassium response followed classical Michaelis-Menten kinetics. The dose-response characteristics of the slow ACh-evoked Cl current displayed a positive cooperativity of at least 3. In spite of this difference, kinetic analysis revealed that these two responses, as well as the fast Cl current response that was characterized earlier (Dascal and Landau 1982), had almost identical apparent equilibrium dissociation constants (0.29–0.39 M), suggesting involvement of a single receptor class. Both K and Cl currents were reduced (to 32–56% of control) by millimolar concentrations of phosphodiesterase (PDE) inhibitors, theophylline and isobutylmethylxanthine. Elevation of extracellular Ca concentration from 1 to 10 mM doubled the K current; depletion of external Ca caused a partial inhibition of this response. The K current was potentiated by 0.1 M 4-phorbol 12,13-dibutyrate (PDBu). Ca-dependence of the ACh-evoked K current resembles that of ACh-evoked Cl currents, described earlier, and suggests mediation by a similar mechanism, i.e. mobilization of Ca from intracellular stores. On the other hand, most of the features described here are in a sharp contrast to those reported for adenosine-evoked, cAMP-mediated slow K current. Thus, we suggest that purinergic and muscarinic receptors inXenopus follicular oocytes are coupled to potassium channels through different molecular mechanisms.Abbreviations ACh acetylcholine - cAMP cyclic adenosine 3,5-monophosphate - cGMP cyclic guanosine 3,5-monophosphate - EGTA ethylenediaminetraacetic acid - Hepes N-2-hydroxyethylpiperazine-N-2-hydroxypropanesulphonic acid - IBMX 3-isobutyl-1-methylxanthine - IP₃ inositol 1,4,5-trisphosphate - PDBu 4-phorbol 12,13-dibutyrate - PDE phosphodiesterase     

Is there a need for lumbar orthosis in mild compression fractures of the thoracolumbar spine?: A retrospective study comparing the radiographic results between early ambulation with and without lumbar orthosis

Compression fractures of the thoracolumbar spine are common in spinal trauma. Most patients are treated with early ambulation. Bracing is an option not always recommended by the treating team. There are no definite recommendations so far regarding the need for lumbar orthosis in this type of injury. The authors retrospectively compared two methods for treating patients with compression as great as 30%: early ambulation with and without lumbar orthosis. The results show that although demographic variables, type of injury, and cause of injury were similar between the groups, neither treatment emerged as superior. Thoracolumbar fractures with compression as much as 30% can be treated with early ambulation and no external support. Close clinical and radiographic follow-up is essential.     

Anxiogenic effects of Sumatriptan in panic disorder: a double-blind, placebo-controlled study.

BACKGROUND: Several lines of evidence point to serotonergic abnormalities in patients with panic disorder (PD). Our goal was to further examine central serotonergic function in panic patients using autonomic and subjective responses to the postsynaptic serotonin 5-HT1D receptor agonist Sumatriptan. METHOD: Using a double-blind, randomized, placebo-controlled design, we assessed autonomic and subjective responses to oral Sumatriptan (100 mg) and placebo in 15 patients with PD, free of medication. Subjective responses were measured using the Hamilton Anxiety Rating Scale (HAM-A), National Institute of Mental Health Anxiety Scale (NIMHA), a modified version of the Panic Symptom Inventory (PI), Hamilton Depression Rating Scale (HAM-D), and Montgomery-Asberg Depression Rating Scale (MADRS). RESULTS: PD patients exhibited significantly enhanced autonomic and subjective responses following challenge with Sumatriptan. We observed an increased pulse rate and augmentation of various parameters measured on different anxiety scales. A constant inclination of aggravation of the measured parameters was detected during the hour post challenge. CONCLUSION: Oral administration of Sumatriptan, a 5-HT1D agonist, has been associated with an anxiogenic effect in PD patients.     

Neoadjuvant chemoradiotherapy for adenocarcinoma of the pancreas: analysis of histopathology and outcome

To examine the histopathologic effect of neoadjuvant therapy and its impact on survival in patients with carcinoma of the pancreas, we retrospectively reviewed the records of 116 patients who underwent resections for pancreatic cancer from 1987 to 2000. Median follow-up of surviving patients was 19 mo(range 4-150 mo). Preoperative chemotherapy was administered in 61 patients (53%) and consisted of 5-fluorouracil/mitomycin C in 35 patients and gemcitabine in 26 patients, given concurrently with external beam radiation (5040 cGy). All resections were performed with curative intent (98 Whipples, 11 total, 6 distal, and 1 central pancreatectomy). Histopathologic examination included an estimation of the amount of fibrosis present in the tumor specimen (expressed as the percentage of fibrosis identified relative to the amount of neoplastic cells present). The mean fibrosis level for the series was 56% (range 5% to 100%). The administration of neoadjuvant therapy resulted in greater fibrosis (73%) than no preoperative treatment (38%) (p = 0.0001). Higher mean fibrosis levels were observed in patients with negative lymph nodes (p = 0.0006) and negative margins (p = 0.05). Factors associated with improved survival(log rank test) included: negative margins (p = 0.001), negative lymph nodes (p = 0.03), and use of neoadjuvant therapy (p = 0.03). Median survival in the neoadjuvant group was 23 mo vs 16 mo without preoperative therapy (p = 0.03). In conclusion, the use of neoadjuvant therapy resulted in a greater degree of fibrosis in the specimen. Patients with negative margins and negative lymph nodes had a greater amount of fibrosis present, and these were significant predictors of improved outcome. Although retrospective,this series suggests an improvement in survival in patients treated with neoadjuvant therapy.     

A balanced 5:1 carbohydrate:protein diet: a new method for supplementing protein to patients with chronic liver disease

BACKGROUND AND AIMS: Protein malnutrition in patients with chronic liver disease contributes to bone and muscle weakness and compromises immune function and survival. In contrast, high-protein diets may induce or exacerbate hepatic encephalopathy. The aim of the present study was to test whether increased amounts of protein, balanced by dietary carbohydrate in a 1:5 ratio, may be given to chronic liver disease patients in order to minimize postprandial increases in plasma amino acid (AA) concentrations. METHODS: Eight patients with chronic liver disease were studied. Each received, in a randomized order, three different diets of 2510 kJ of either high protein (37:50:28, carbohydrate:protein:fat), high carbohydrate (126:10:6) or a balanced 5:1 carbohydrate:protein diet (105:21:11). All patients were followed for plasma AA, glucose and insulin levels, as well as for cognitive and behavioral changes. RESULTS: Following the high protein diet, AA concentrations were significantly increased. In contrast, after the balanced diet, AA levels were practically constant enabled. All diets was well tolerated and no cognitive or behavioral changes appeared. CONCLUSION: The administration of a balanced 5:1 carbohydrate:protein diet may enable patients with chronic liver disease to tolerate increased amounts of dietary protein, without altering plasma amino acid concentrations.