Search

Subtypes I, II and III of sodium channel alpha-subunit mRNAs were analyzed in adult rat brain areas after kainate-induced seizures. Tissue samples were microdissected from occipital neocortex, CA1 and CA3 hippocampus areas and dentate gyrus. Three reverse transcriptase-polymerase chain reaction (RT-PCR) protocols were undertaken to amplify these mRNAs. Amplification products were then distinguished after digestion by restriction enzymes, electrophoresis separation and densitometric analysis of gel profiles. PCR 1 evidenced the relative percentage of mRNAs I, II and III as well as neonatal II and III subtype isoforms, which resulted from an alternative splicing. PCR 2 and 3 were performed to focus on the neonatal vs. adult ratio in II and III subtypes, respectively. Seizures were shown to induce an increase in both neonatal subtypes, which suggested an alteration at the splicing level. These changes exhibited a peculiar brain regional distribution, the maximal effect being observed in dentate gyrus and hippocampus CA1 area. In situ hybridization experiments, using a digoxigenin-labeled oligonucleotide probe-specific for neonatal II and III mRNAs, confirmed this increase in neonatal mRNA subtypes. These changes were transient, reaching a maximum 6 h after drug injection, then disappearing between 12 and 48 h. They were prevented by a pre-treatment of animals by MK-801, a non-competitive antagonist of NMDA receptors. This work, thus, suggested that KA-induced seizures can be accompanied by transient alteration in the splicing pattern of sodium channel alpha-subunit mRNAs which resulted in an increase in expression of their neonatal isoforms within localized areas of adult rat brain.

The intricate regulation of Spot 14 expression in rat lipogenic tissues has provided a useful tool in studying nutritional and hormonal factors involved in transcription. To gain insight into its function and its possible involvement in human lipid disorders, we cloned human and mouse Spot 14 genes that shared with the rat gene a strong homology concerning the deduced amino acid sequence (81 and 94%, respectively) as well as the promoter region. The mouse promoter was characterized by transfection studies, while quantitative RT-PCR and in situ hybridization experiments showed that Spot 14 is expressed in human liver and, at a high level, in multiple symmetric lipomatosis nodules.

Objective: To evaluate the GH-releasing hormone (GH-RH)-induced response of GH in patients affected by hypothalamic amenorrhea., Design: Patients affected by weight-loss-related hypothalamic amenorrhea (n = 28) were studied and compared with 20 healthy controls. Among patients with weight-loss amenorrhea, both hypogonadotropic and normogonadotropic conditions were present. All subjects underwent a GH-RH test (GEREF, Sereno, Rome, Italy) (1 microgram/kg body weight IV). Plasma GH concentrations were determined using commercially available RIAs. Also, in selected samples insulin-like growth factor-I (IGF-I) levels were measured., Results: Basal plasma IGF-I levels as well as body mass index (BMI) were lower in amenorrheic patients than in healthy controls. No significant correlation was found between BMI and IGF-I or E2 plasma levels or between LH and IGF-I plasma levels. The basal GH plasma levels were comparable in all groups of subjects. The GH-RH--induced GH response evaluated as maximal release and as area under the curve (AUC) was higher in amenorrheic patients than in control subjects., Conclusions: The amenorrheic condition associated with reduced BMI changes the GH-RH--induced GH response in hypothalamic amenorrhea, supporting a GH and a IGF-I disregulation in weight-loss--related amenorrhea.

Objective: To evaluate whether the efficacy of naltrexone administration in patients with hypothalamic amenorrhea correlates to the response to an acute naloxone test., Design: Thirty patients with hypothalamic amenorrhea associated with weight loss were studied. After naloxone test (4 mg in bolus IV) patients were divided into two groups: group A, nonresponsive (n = 15) and group B, responsive (n = 15). Group A underwent two cycles of hormonal replacement therapy with E2 patches and medroxyprogesterone acetate. Then all patients were administered naltrexone at the dosage 50 mg/d orally for 6 months. A third group of 10 amenorrheic patients were treated with oral placebo with the same schedule., Results: Plasma gonadal steroid levels increased in all patients and in 24 of 30 patients the menstrual bleeding occurred within 90 days from the beginning of treatment. After 6 months from naltrexone discontinuation, 18 of 24 patients still showed the occurrence of menstrual cycles. Luteinizing hormone plasma levels and LH pulse amplitude increased after 3 months of treatment and remained unchanged 6 months after naltrexone suspension. Plasma FSH levels did not show any change in any patient. The body mass index increased after 3 months in all patients who menstruated. Patients treated with placebo did not show any significant change in gonadotropins and gonadal steroid plasma levels., Conclusions: The present study supports the efficacy of naltrexone therapy for patients with hypothalamic amenorrhea either responsive or nonresponsive to naloxone test.

Bile salt-dependent lipase (BSDL), an enzyme normally found in human pancreatic secretions is a 100 kDa glycoprotein. A BSDL-specific 477 bp cDNA probe was prepared by performing polymerase chain reaction experiments. This cDNA was used to probe mRNAs extracted from human pancreatic tissue and tumoral cell lines. Two mRNAs were detected in normal human pancreas at 2.2 and 1.3 to 1.5 kb. In human pancreatic tumoral cells, mRNAs encoding for the BSDL were detected using in situ hybridization, and proteins with an M(r) of 46,000 to 48,000 were translated into an in vitro system using mRNAs extracted from these cells. Using an immunoprecipitation procedure, we observed here that the specific BSDL polyclonal antibodies recognized three proteins of 100 +/- 5 (p100), 46 +/- 2 (p46) and 22.7 +/- 1.2 (p23) kDa, respectively in the soluble extracts of normal adult human pancreas. The p100 protein was probably the glycosylated product resulting from the translation of the 2.2 kb transcript. The p46 protein, which electrophoresed as a doublet was the main component immunoprecipitated from extract of a differentiated human pancreatic adenocarcinoma as well as from the extracts of two pancreatic cell lines, BxPC-3 and SOJ-6. In addition, the p46 immunoform of the BSDL was detected in cell-free medium from SOJ-6 cell line and its expression was found to be correlated with the secretion of an esterolytic activity on 4-nitrophenyl caproate, whereas the BxPC-3 cell line neither secreted the p46 nor showed any esterolytic activity on this substrate. The p46 may be either a short variant of BSDL resulting from the translation of the 1.3 to 1.5 kb transcript or a protein structurally related to the enzyme. The p46 doublet immunoform was detected in the human pancreatic secretion.

The intrinsic characteristics of LH and prolactin (PRL) episodic secretion were evaluated in a group of 18 children (8M and 10F). The children were divided into two groups according to the Tanner stage: Group A (Tanner < or = 1, N = 7, 3M and 4F, 6-10 years of age) and group B (Tanner 2-3, N = 11, 5M and 6F, 9-11 years of age). A pulsatility study of 4 h, sampling every 10 min, was carried out in all children. LH and PRL plasma levels were assayed by IFMA and RIA respectively. LH and PRL secretory episodes were then identified on plasma determinations using the program DETECT. Instantaneous secretory rates (ISR) were then computed for both LH and PRL using the specific algorithm within the DETECT program. Plasma LH levels were different between the two groups of children. Group A children showed undetectable LH plasma levels (below the minimal detectable dose of 0.1 mIU/ml), while group B demonstrated LH plasma levels in the normal range of values for age and sexual development (1.5 +/- 0.3 mIU/ml, mean +/- SEM). LH pulse frequency for group B was 3.2 +/- 0.4 peaks/4 h. No significant differences in mean plasma PRL levels, pulse frequency and pulse amplitude were observed between the two groups of children. Computation of ISR for LH (group B only) and PRL (both groups) identified the intrinsic episodic characteristics of the two hormones. No significant differences in LH and PRL pulse frequencies were observed when comparing the results estimated on ISR with those estimated on plasma concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

Amenorrhea associated with weight loss is characterized by several neuroendocrine aberrations. The aim of the present study was to define the characteristics of episodic secretion of prolactin in women with weight loss-related amenorrhea. To evaluate whether hypoestrogenism was responsible for the changes in prolactin secretion in amenorrheic women, the pulsatile secretory pattern was also studied during hormone replacement therapy (HRT). Fifteen patients were studied by blood sampling every 10 min for 8 h. Four normally cycling women were studied as a reference group, during both the midfollicular and midluteal phases. Mean plasma prolactin levels and pulse amplitude were lower in amenorrheic patients than in controls. The prolactin pulse frequency was higher than that in controls during the follicular phase, but lower than controls during the luteal phase of the menstrual cycle. During HRT, mean plasma prolactin levels significantly increased, pulse frequency decreased and pulse amplitude significantly increased both during estradiol and estradiol plus medroxyprogesterone acetate administration. In conclusion, in amenorrhea associated with weight loss the chronobiological organization of prolactin secretion is deranged, both as a result of a neuroendocrine alteration and as a result of low plasma levels of gonadal steroids.

Most recently reported methods to select early hematopoietic cells basically rely on the depletion of committed progenitors. This task is generally accomplished by laborious procedures, which are sometimes difficult to reproduce. To simplify the selection method, we took advantage of the expression of the transferrin receptor (CD71) by proliferating committed progenitors and the lack of CD71 on noncycling immature progenitors. A monoclonal antibody (MAB) reactive with CD71 has been conjugated to the Saponaria officinalis seed ribosome-inactivating protein (SO6). The immunotoxin (IT) complex was used at increasing concentrations on normal non-phagocytizing bone marrow cells. A complete and reproducible killing effect on myeloid (colony-forming unit-granulocyte/macrophage [CFU-GM]) and erythroid (burst-forming unit-erythroid [BFU-E]) progenitors was observed for IT concentrations of 1 x 10(-7) M. Unconjugated SO6 or anti-CD71 MAB had no effect on cell growth and viability. IT-resistant cells were able to generate CFU-GM after 7, 14, and 21 days of suspension culture in the presence of 5637 CM. Maximal CFU-GM values were obtained at day 21 and nearly approached the pretreatment values (mean 2587 vs. 3877 CFU-GM/mL). Growth factor enhancement of CFU-GM yield was obtained only by stem cell factor (SCF) at day 7; SCF, as well as granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3), had an enhancing effect at days 14 and 21. IT toxicity on highly immature progenitors was ruled out by evaluating the growth of long-term culture-initiating cells (LTC-IC) from IT-treated cultures. LTC-IC frequency was found to be 1 out of 1506 seeded cells, which is within the range of normal untreated BM cells. In conclusion, anti-CD71 IT allows a simple and complete depletion of committed progenitors while sparing immature hematopoietic cells. The high CD71 expression by leukemic cells makes the procedure potentially suitable for in vitro purging.

Objective: To define the characteristics of spontaneous GH episodic secretion and the modulatory role of gonadal steroids in patients with hypothalamic amenorrhea associated with weight loss., Design: Women were studied for 8 hours, sampling every 10 minutes, and plasma GH levels were measured by RIA., Subjects: Fifteen patients with weight-loss-related amenorrhea were studied in baseline conditions. Five out of 15 patients underwent two cycles of hormonal replacement therapy with E2 patches (100 micrograms every 3 days for 24 days) and medroxyprogesterone acetate (MPA) (10 mg/d, from day 12 to day 24). On the second cycle of therapy, the pulsatility study was repeated twice: after only estrogen (day 11) and after E2 plus progestin (day 22). Four normally cycling women were studied as a reference group during midfollicular and midluteal phases., Results: Amenorrheic patients showed mean plasma GH levels similar to healthy women during the follicular phase but significantly lower than those observed during the luteal phase. GH pulse frequency was higher in patients than in controls, whereas pulse amplitude was comparable with the follicular phase but lower during the luteal phase. During the hormonal replacement therapy, when only E2 was administered, GH pulse frequency decreased, whereas GH integrated plasma concentrations and GH pulse amplitude increased significantly. After MPA and E2 administration, GH pulse amplitude and GH plasma levels decreased, which was similar to pretreatment condition., Conclusions: The present study demonstrated that in amenorrhea associated with weight loss the frequency of GH episodic release is significantly higher than in normally cycling women. Moreover, a different modulatory role of estrogen (increased amplitude) and P (decreased amplitude) on the episodic release of GH in amenorrheic women undergoing a replacement treatment was shown by the present data.

Challenging intact erythrocytes from naive rats with ethanol resulted in dose-dependent decreases in rates of acylation of phosphatidylcholine and phosphatidylethanolamine. In erythrocytes from ethanol-treated animals, the responses were of lesser magnitude, indicating a lower sensitivity to ethanol. This relative resistance, typical of the state of tolerance, was not associated with increased baseline rates of acylation of PC and PE, nor with changes in fatty acid specificity of acylation reactions. Taken together, the data suggest that (1) intact rat erythrocytes represent a reliable and easily reproducible model for studying biochemical correlates of the adaptive response to ethanol; (2) phospholipid acylation reactions are implicated in the initial sensitivity and subsequent acquisition of tolerance to ethanol in membrane erythrocytes; (3) on the basis of the measured acylation reactions, rat erythrocytes appear to develop tolerance, but not dependence, to ethanol.

We examined the effects of the in vivo administration of ethanol on lipolytic activities assayed in rat post-heparin heart effluents, that hydrolyse tri-, di- and monoacylglycerol. Properties of triacylglycerol lipase (TAGL) are typical of lipoprotein lipase (LPL) whereas diacylglycerol (DAGL) and monoacylglycerol (MAGL) lipase activities hydrolyse sequentially the products of LPL action. After 15 days of ethanol intake, TAGL, DAGL and MAGL activities in post-heparin heart effluents were decreased respectively by 25, 38 and 22%; after 30 days, the decreases amounted to 81, 79 and 71%. After 30 days, but not after 15 days, ethanol increased the levels of triacylglycerol in plasma. Ethanol intake concomitantly decreased TAGL and DAGL activities in post-heparin effluents and in heart tissue extracts, whereas MAGL activity was decreased only in the latter extracts. We conclude that ethanol intake causes a marked impairment in heart LPL and in two closely-related heparin-releasable activities, seemingly by altering the production of a catalytically active enzyme. A distinct heparin-unreleasable MAGL appears to exist in heart, that could be ethanol-insensitive. Overall, the results suggest that a LPL-related alteration in fatty acid supply could contribute to the toxicity of ethanol in heart.

1. Triacylglycerol in erythrocytes from normal human subjects was estimated to average 2.7 +/- 0.7 nmol/10(10) cells, equivalent to 0.07% of total lipids or 0.3% of neutral lipids. 2. The specific activity of triacylglycerol labelling attained by incubating intact erythrocytes with [3H]oleic acid was 10 nmol/mumol, a value 20-fold higher than that of the highest labelled phospholipid, sphingomyelin; as isolated by ultracentrifugation over a density gradient, the youngest erythrocytes exhibited a labelling rate 10-fold higher than that of older cells. 3. The triacylglycerol content was not modified in erythrocytes from chronic alcoholics, whereas the mean rate of triacylglycerol labelling was 31% (P less than 0.05) higher than that of control subjects, and did not normalize 4 weeks after alcohol withdrawal. 4. These results indicate that triacylglycerol, although a quantitatively minor component, is one of the most active metabolites in the lipid matrix of the human erythrocyte membrane and appears to be implicated in the membrane response to antagonistic agents.

The present paper reports the results of an immunohistochemical study of 6 cases of monomorphic (2 clear cell, 4 basal cell) and 12 cases of pleomorphic adenomas in the parotid gland. The surgical specimens, embedded in paraffin, were tested by antisera vs. epithelial cell markers with epithelial membrane antigen (EMA) and cytokeratins (CK). They were also tested vs. myoepithelial and non-epithelial markers with S-100 protein and Glial Fibrillary Acidic Protein (GFAP). In pleomorphic adenomas, positive cells were found for all the tested antigens. Positive cell distribution showed a marked variability from case to case and even within each area, depending on cell morphology. In particular, areas showing tubular structures were constantly EMA-positive and CK-positive in more than 50% of the cases. In epithelioid areas EMA was negative while many cells were variably positive for CK, S-100 and GFAP. At times, the same cell proved positive to two different antigens. In those fuso-stellate (myxoid) cell areas considered to be of myoepithelial origin, cells were slightly CK-positive and widely GFAP and S-100 positive. In basal cell adenomas, widespread CK positivity was uniformly present while rare, focal positivity was evidenced for EMA and widespread non reactivity was found for S-100 and GFAP. In clear cell adenomas focal CK expression was found in the tubular lumina while clear cells, of myoepithelial origin, were steadily S-100 positive and GFAP-negative. It was noted that a sort of S-100/GFAP positive myoepithelial cell with fuso-stellate morphology was present in pleomorphic adenomas.(ABSTRACT TRUNCATED AT 250 WORDS)

Several neuroendocrine disregulations have been demonstrated in patients with hypothalamic amenorrhea, but a definite therapeutic strategy has not yet been found. Since acetyl-l-carnitine (ALC) has been reported to have a specific effect on central cholinergic, serotoninergic, dopaminergic and opioidergic systems, 20 patients with hypothalamic amenorrhea were treated with ALC (2 g/day, per os). Both the clinical efficacy and the endocrine parameters were evaluated after 6 months. The patients were subdivided in two groups according to their LH plasma levels: A) hypogonadotropic: 10 subjects with plasma LH less than 3 mIU/ml, and B) normogonadotropic: 10 subjects with plasma LH greater than 3 mIU/ml. All subjects underwent: 1) a pulsatility study (4 h sampling every 10 min), 2) GnRH test (two bolus injections of 10 micrograms at time 0 and +120), 3) TRH test (200 micrograms). These parameters were evaluated before and after 6 months of ALC administration. The occurrence of a spontaneous menstruation was observed in 6 out of 10 hypogonadotropinemic and in 4 out of 10 normogonadotropinemic patients. Menstrual bleeding occurred between the 3rd and the 6th month of therapy. Major hormonal changes after ALC administration were observed in the hypogonadotropic subjects. They showed a significant increase in baseline plasma LH levels (from 0.9 +/- 0.1 to 3.5 +/- 0.7 mIU/ml, p less than 0.05) (mean +/- SEM), a significant increase in LH pulse amplitude (p less than 0.01) with no changes in LH pulse frequency, and a significantly increased response of LH to the latter GnRH bolus during the GnRH test. Hypogonadotropic patients also showed a significant increase in both estradiol (from 18.8 +/- 2.5 to 48 +/- 3.3 pg/ml, p less than 0.05) and PRL (from 6 +/- 1 to 11.4 +/- 1.7 ng/ml, p less than 0.05). No significant differences were observed in the hormonal parameters of normogonadotropic patients after 6 months of ALC therapy.(ABSTRACT TRUNCATED AT 250 WORDS)

The lymphocyte subsets in 67 cases of bone marrow benign or reactive lymphoid nodules (LN) and 23 cases of nodular involvement by B malignant lymphoma (B-ML) have been immunohistochemically characterized on paraffin embedded trephine biopsies utilizing a panel of 10 monoclonal antibodies. LCA was positive in 90% LN and B-ML lymphocytes; LN2, MB2 stained more than 50% LN and B-ML lymphocytes; MT2 stained more than 50% LN and less than 50% B-ML lymphocytes; UCHL1 stained the 20% of LN lymphocytes; LN1 stained only B cells of the rare germinal centers; MT1 only myeloid cells, L26 only plasma cells. DF-T1 and Leu 22 failed to stain LN or B-ML lymphocytes. While anti-T lymphocyte antibodies reacted inconstantly with lymphoid cells in decalcified and embedded specimens, LCA and some anti-B lymphocyte antibodies gave constantly reproducible results for bone marrow LN and B-ML. They permitted an easy recognition and exact evaluation of the size of LN, the identification of scattered B cells, the detection of the residual or minimal involvement by B-ML and the exact burden of the invasion, but could not allow a convincing differential diagnosis between LN and small cell B-ML nodular involvement.

Monoester lipase activity was assayed by a radiochemical assay in the red blood cells (RBC) from 50 chronic alcoholic patients within 48 hr after discontinuation of chronic alcohol intake and from 40 nonalcoholic control subjects. The mean value of lipase activity was increased to 1213 +/- 229 milliunits/10(12) RBC in the alcoholics as compared with a value of 997 +/- 120 milliunits/10(12) RBC in the controls (p less than 0.001). The lipase increase was associated with increased values of the mean cellular volume of RBC. A subgroup (64%) of 32 alcoholic subjects with macrocytosis (mean cellular volume greater than 96 femtoliters) showed the highest mean lipase activity (1276 +/- 224 milliunits/10(12) RBC) as compared with a value of 1101 +/- 196 milliunits/10(12) RBC in the normocytic alcoholic subgroup (p less than 0.05). This latter subgroup had a mean value higher (p less than 0.05) than that in the control group. The relationship between values of mean cellular volume and lipase activity was not of predictive value in individual cases. The enzymatic increase was not related to a direct effect of alcohol on the intact RBC. It is postulated that this alteration might result from changes in the chemical and/or physical state of the plasma membrane induced by ethanol during RBC formation. In any event, the increased lipolytic activity of RBC represents a new biological characteristic of alcoholic subjects. Its determination might represent a noninvasive way of evaluating the influence of alcohol on a tissue parameter.

The composition and metabolism of erythrocyte lipids were studied in 10 chronic alcoholic patients within 48 hr after discontinuation of alcohol intake and in 10 nonalcoholic control subjects. Chronic alcoholism produced no change in contents of cholesterol, total phospholipids, and proportions of phosphatidylcholine and phosphatidylethanolamine in erythrocyte phospholipids. The mean values of the rates of acylation of phosphatidylcholine and phosphatidylethanolamine with oleic acid were increased respectively by 59% (p less than 0.001) and 38% (p less than 0.05) as compared with the controls. There was no correlation between acylation rates and mean cellular volumes. Increases in acylation rates normalized over several weeks after alcohol withdrawal and were not related to a direct effect of alcohol on the intact erythrocyte, suggesting that these alterations result from ethanol-induced changes in the membrane during erythrocyte formation. The increased rates of acylation might modify the remodeling of the lipid matrix and thereby the membrane function.