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Objectives: To evaluate the diagnostic accuracy of liver fibrosis tests and its influencing factors in a meta-analysis with individual data., Design and Methods: Four independent centers provided four blood tests and Metavir staging from 825 patients with chronic hepatitis C., Results: FibroMeter AUROC (0.840) for significant fibrosis was superior to those of Fibrotest (0.803, p=0.049), APRI (0.789, p=0.001) and Hepascore (0.781, p<0.001). The misclassification rate was lower for FibroMeter (23%) than for Fibrotest and Hepascore (both 28%, p<0.001). The variation in the diagnostic cut-offs of tests among centers, reflecting the overall reproducibility, was: FibroMeter: 4.2%, APRI: 24.0%, Fibrotest: 24.2%, Hepascore: 35.0%. Accordingly, the proportion of patients diagnosed with significant fibrosis changed: FibroMeter: 0.8%, Hepascore: 2.4% (p=0.02 vs FibroMeter), Fibrotest: 5.8% (p<10(-3)), APRI: 18.2% (p<10(-3))., Conclusions: This study on clinical applicability shows significant differences in diagnostic accuracy, inter-center reproducibility, and robustness of biomarkers to changes in population characteristics between blood tests.

Objective: Measurement of thyroglobulin in the washout of lymph node (LN) fine needle aspirates is recommended in the follow-up of patients with differentiated thyroid cancer (DTC). The significance of low fine needle aspirates thyroglobin (FNATg) levels remains a question, which we addressed., Method: Prospective study comparing FNATg with FNA cytology. Exploration of 34 DTC patients (53 cervical LNs), 26 non-thyroidectomized patients with a thyroid-unrelated cervical mass (negative controls) and 13 with 21 thyroid nodules (positive controls). The 12 DTC patients (19 LNs) with a malignant FNA cytology and/or high FNATg level received LN surgery (11 patients) or I(131)-iodine treatment (1 patient) and the outcome measure was pathological or scintigraphic evidence of DTC LN metastasis., Results: All 26 negative controls showed FNATg <1 ng/FNA and all 21 positive controls showed high levels of FNATg (127-210,000 ng/FNA, median 38,000). Among DTC patients in 25 LNs with a benign FNA cytology, FNATg was undetectable in 24 and low in 1 (6 ng/FNA); in 19 LNs with a malignant FNA cytology, FNATg was high in 17 (80-140,000 ng/FNA, median 7174 ng/FNA) and low in 2 (6.6 and 7.1 ng/FNA), which proved to be low Tg immunostaining oncocytic DTC metastasis; in 9 LNs with a non-informative cytology, FNATg was undetectable in 8 but 11,825 ng/FNA in 1, which proved a DTC metastasis. Measurement of FNA albumin demonstrated that contamination of FNA by serum proteins was negligible., Conclusion: Low FNATg levels can indicate a DTC metastasis. It cannot be related to clinically relevant levels of serum Tg.

Patients affected by the multiple endocrine neoplasia type I syndrome (MEN1) display a high incidence of pituitary adenomas, though it is still unknown whether these pituitary tumors have specific pathologic features that would distinguish them from sporadic pituitary adenomas. Pituitary tissue specimens of 77 MEN1 patients from the GTE (Groupe d'étude des Tumeurs Endocrines) register were compared with unselected 2509 non-MEN1 sporadic pituitary tumors and also to a control subgroup of 296 cases, where 1 MEN1 tumor was matched with 4 sporadic tumors of the same hormonal immunoprofile. Sex, age, size, and invasiveness of tumors, and menin gene mutations were documented. Histologic analysis took into account 33 items, including immunocytochemical data, the proliferative marker Ki-67, and an examination of the juxtatumoral pituitary. MEN1 tumors were significantly larger and more often invasive by histology. MEN1 patients with large pituitary tumors (grade IV) were younger than non-MEN1 patients. MEN1 tumors had no other characteristic histologic features and no predominance of any one hormone producing subtype. However, plurihormonal adenomas versus monohormonal and nonimmunoreactive adenomas were more frequent in MEN1 tumors (39%) than in the control non-MEN1 group (P = 0.001). Especially, the growth hormone and prolactin plurihormonality with unusual association with follicle-stimulating hormone, luteinizing hormone, or adrenocorticotropic hormone was more frequent in MEN1 tumors. In addition, multiple adenomas were significantly more frequent (4% vs. 0.1%; P < 0.0001), especially prolactin-adrenocorticotropic hormone. Somatotroph hyperplasia, with or without a microadenoma was found in only 3 MEN1 patients, with growth hormone-releasing hormone hypersecretion by a pancreatic tumor in 2 of them. All types of mutation were observed, including frameshifts, nonsenses, missenses, and 1 case of germline MEN1 encompassing large deletion, strongly suggesting the absence of any phenotype-genotype correlation.

Adrenocortical carcinoma (AC) mixed with a sarcoma or sarcoma-like component is exceptional, and only six cases have been detailed in the literature, three including osteo-, chondro-, or rhabdomyosarcoma components, and three others only showing a malignant spindle cell component. These histological subtypes, respectively called adrenal carcinosarcomas and sarcomatoid AC, represent poorly differentiated and extremely aggressive forms of carcinoma, with locoregional recurrence and metastases rapidly arising from the sarcomatous or sarcomatoid component, and death occurring in a few months. We report a case of AC in a 31-year-old man presenting as a nonfunctional tumor, with a histological biphasic pattern combining few areas of differentiated AC and extensive areas of sarcomatoid spindle cell proliferation. The patient died 3 months of locoregional and distant recurrences after surgery despite apparently total tumor resection and VP16-cisplatinum chemotherapy. This case underlines the necessity to identify and isolate these carcinoma's subtypes with worse prognosis and the difficulties to distinguish them from metastatic carcinomas and retroperitoneal sarcomas, in relation to the particular adrenal cortex immunoprofile. According to the World Health Organization principles of terminology, we suggest these tumors be collectively classified as "adrenal sarcomatoid carcinomas," a designation that tends to unify all carcinomas with "pleomorphic, sarcomatoid, or sarcomatous elements."

Rationale: Intermittent hypoxia, a hallmark of sleep apnea, is a major factor for hypertension and impaired vasoreactivity., Objectives: To examine the temporal occurrence of these two outcomes in order to provide insight into mechanisms and early cardiovascular disease identification., Methods: Functional and structural cardiovascular alterations were assessed in C57BL6 mice exposed to intermittent hypoxia (21-4% Fi(O(2)), 30-s cycle, 8 h/d) or air for up to 35 days. Blood pressure, heart rate, and urinary catecholamines were measured at Days 1 and 14. Hindquarter vasoreactivity was assessed at Days 14 and 35, including vasoconstriction to norepinephrine, endothelium-, and non-endothelium-dependent vasodilation. Aorta, heart, and hindquarter skeletal muscles were immunostained for vascular markers PECAM-1 and collagen IV., Measurements and Main Results: Hemodynamic alterations occurred from Day 1, characterized by blood pressure surges with bradytachyarrhythmia driven by cyclic hypoxia. At Day 14, blood pressure at normoxia was elevated, with predominant diastolic increase. With hypoxia, vasopressive catecholamines were elevated, blood pressure surged with a lower hypoxic threshold, whereas heart rate fluctuations decreased. Histologic alterations started from Day 14, with decreased endothelial PECAM-1 expression in descending aorta and left heart. Impaired peripheral vasoreactivity occurred at Day 35, including hypervasoconstriction to norepinephrine secondary to sympathetic hyperactivity, without changes in pre- and postsynaptic alpha-adrenoceptors or in endothelium- and non-endothelium-dependent vasodilation., Conclusions: Intermittent hypoxia induces sequential cardiovascular events suggesting increased chemoreflex and depressed baroreflex, resulting in sympathoadrenal hyperactivity, early hemodynamic alterations with proximal histologic remodeling, and delayed changes in peripheral vasoreactivity. Such early alterations before overt cardiovascular disease strengthen the need for identifying at-risk individuals for systematic treatment.

Cushing's syndrome due to ACTH-independent macronodular adrenal hyperplasia (AIMAH) can be associated with abnormal responses of aberrantly expressed adrenocortical receptors. This study aimed to characterize in vitro the pathophysiology of hypercortisolism in a beta-blocker-sensitive Cushing's syndrome due to AIMAH. Cortisol secretion profile under aberrant receptors stimulation revealed hyperresponsiveness to salbutamol (beta2-adrenoceptor agonist), cisapride (5-HT4 receptor agonist), and vasopressin in AIMAH cultured cells, but not in normal adrenocortical cells. By RT-PCR, AIMAH tissues revealed beta2-adrenoceptor overexpression rather than ectopical expression. MC2R expression was similar in both AIMAH and normal adrenocortical tissues. Curiously, cortisol levels of AIMAH cells under basal condition were 15-fold higher than those of control cells and were not responsive to ACTH. Analysis of culture medium from AIMAH cells could detect the presence of ACTH, which was immunohistochemically confirmed. Finally, the present study of AIMAH cells has identified: a) cortisol hyperresponsiveness to catecholamines, 5-HT4 and vasopressin in vitro, in agreement with clinical screening tests; b) abnormal expression of beta2-adrenoceptors in some areas of the hyperplastic adrenal tissue; c) autocrine loop of ACTH production. Altogether, the demonstration of aberrant responses to hormonal receptors and autocrine hormone production in the same tissue supports the assumption of multiple molecular alterations in adrenal macronodular hyperplasia.

Unlabelled: CD8(+) T cells represent a sizable component of the liver inflammatory infiltrate in chronic hepatitis C and are thought to contribute to immune-mediated tissue injury. Because chronic stimulation may promote the expression by CD8(+) T cells of distinct human leukocyte antigen class I-specific natural killer cell receptors (NKRs) susceptible to both inhibiting effector functions and promoting cell survival, we examined the distribution and characteristics of CD8(+) T cells with such receptors in chronic hepatitis C patients. NKR CD8(+) T cells were detectable in liver and peripheral blood from hepatitis C virus (HCV)-infected patients but were not major subsets. However, the frequency of NKG2A(+) CD8(+) in the liver and in a lesser extent in the peripheral blood was positively correlated to histological activity in HCV-infected patients. No such correlation was found with KIR(+) T cells in liver in HCV-infected patients and with the both NKR CD8(+) T cells in hepatitis B virus (HBV) infected patients. Circulating CD8(+) T cells expressing KIRs exhibited phenotypic features of memory T cells with exacerbated expression of the senescence marker CD57 in patients. NKG2A(+)CD8(+) T cells were committed T cells that appeared less differentiated than KIR(+)CD8(+) T cells. In HCV-infected patients, their content in perforin was low and similar to that observed in NKG2A(-)CD8(+) T cells; this scenario was not observed in healthy subjects and HBV-infected patients. Both NKG2A and KIRs could inhibit the response of HCV-specific CD8(+) T cells ex vivo., Conclusion: These results support the concept that an accumulation in the liver parenchyma of NKR(+)CD8(+) T cells that have functional alterations could be responsible for liver lesions. They provide novel insights into the complexity of liver-infiltrating CD8(+) T cells in chronic hepatitis C and reveal that distinct subsets of antigen-experienced CD8(+) T cells are differentially sensitive to the pervasive influence of HCV.

Glyoxalase I (GloI) catalyzes the glutathione-dependent conversion of 2-oxoaldehydes to S-2-hydroxyacylglutathione derivatives. Studies on GloI from diverse organisms such as man, bacteria, yeast, and different parasites show striking differences among these potentially isofunctional enzymes as far as metal content and the number of active sites per subunit are concerned. So far, it is not known whether this structural variability is linked to catalytic or regulatory features in vivo. Here we show that recombinant GloI from the malaria parasite Plasmodium falciparum has a high- and a low-affinity binding site for the diastereomeric hemithioacetals formed by addition of glutathione to methylglyoxal. Both active sites of the monomeric enzyme are functional and have similar k(cat)(app) values. Proteolytic susceptibility studies and detailed analyses of the steady-state kinetics of active-site mutants suggest that both reaction centers can adopt two discrete conformations and are allosterically coupled. As a result of the positive homotropic allosteric coupling, P. falciparum GloI has an increased affinity at low substrate concentrations and an increased activity at higher substrate concentrations. This could also be the case for GloI from yeast and other organisms. Potential physiologically relevant differences between monomeric GloI and homodimeric GloI are discussed. Our results provide a strong basis for drug development strategies and significantly enhance our understanding of GloI kinetics and structure-function relationships. Furthermore, they extend the current knowledge on allosteric regulation of monomeric proteins in general.

Background/aims: Non-invasive markers of liver fibrosis have recently been developed as an alternative to liver biopsy. The aim of this study was to compare the diagnostic performance of 6 scores (MP3, Fibrotest, Fibrometer, Hepascore, Forns' score and APRI)., Methods: We studied 180 chronic hepatitis C patients. Liver fibrosis was staged according to the METAVIR scoring system., Results: Overall diagnostic performance of scores determined by AUROCs ranged from 0.86 for Fibrometer to 0.78 for Forns' score (NS) for discriminating F0F1 versus F2F3F4. For discriminating F0F1F2 versus F3F4, AUROCs ranged from 0.91 for Fibrometer to 0.78 for Forns' score (p<0.02). Significant or extensive fibrosis was predicted in 10-86% of patients with positive predictive value (PPV) ranging from 55% to 94%. Using logistic regression, statistical independence was demonstrated for MP3, Fibrotest and APRI. Diagnostic performance of paired-combination scores was then evaluated. The best combinations could select one-third of patients for whom either absence of significant fibrosis or presence of extensive fibrosis could be predicted with more than 90% of certainty., Conclusions: Current non-invasive scores give reliable information on liver fibrosis in one-third of chronic hepatitis C patients, especially when used in combination.

Biallelic somatic mutations of TCF1 coding for hepatocyte nuclear factor 1alpha (HNF1alpha) are found in 50% of the hepatocellular adenoma (HCA) cases usually associated with oral contraception. In rare cases, HNF1alpha germ line mutations could also predispose to familial adenomatosis. In order to identify new genetic factors predisposing to HNF1alpha-mutated HCA, we searched for mutations in genes involved in the metabolism of estrogen. For 10 genes (CYP1A1, CYP1A2, CYP3A4, CYP3A5, COMT, UGT2B7, NQO1, GSTM1, GSTP1, and GSTT1), we did not find mutations nor differences in the allele distribution among 32 women presenting HNF1alpha-mutated adenomas compared with 58 controls. In contrast, we identified a CYP1B1 germ line heterozygous mutation in 4 of 32 women presenting HNF1alpha-mutated adenomas compared with none in 58 controls. We confirmed these results with the identification of four additional CYP1B1 mutations in a second series of 26 cases. No mutations were found in the control group, which was extended to 98 individuals, and only a known rare genetic variant was observed in two controls (P = 0.0003). We did an ethoxyresorufin O-deethylase assay to evaluate the functional consequence of the CYP1B1 mutations. We found reduced enzymatic activity in each CYP1B1 variant. In addition, an E229K CYP1B1 mutation was found in a woman with a germ line HNF1alpha mutation in a familial adenomatosis context. In this large family, all three patients with adenomatosis bore both HNF1 and CYP1B1 germ line mutations. In conclusion, our data suggested that CYP1B1 germ line-inactivating mutations might increase the incidence of HCA in women with HNF1alpha mutations.

Objective: Genetic diagnosis available since 1993 established germinal mutations of the RET proto-oncogene at codon 634 as the main cause of inherited medullary thyroid carcinoma (MTC). International guidelines established in 1999 recommend that children with such mutations undergo a total thyroidectomy before age 5, with unspecified cervical neck dissection. Since 1993, only 41 of 275 thyroidectomies reported in RET 634 children were performed before age 5 (15%). The aim of this study was to evaluate the implementation of these guidelines in a single centre., Design and Patients: Genetic diagnosis was proposed to the parents of all eight children born after 1992 from two RET 634 families. Total thyroidectomy was proposed before age 5 if the child carried a mutation., Results: Genetic diagnosis was performed in all the children (aged 1-3) and thyroidectomy in the five who carried a mutation (aged 2-5). Cervical lymph node dissection varied from lymphadenectomy of central and lateral compartments in the eldest child to pickings in the youngest. There was no permanent hypoparathyroidism or recurrent nerve paralysis. C-cell hyperplasia, medullary thyroid carcinoma and lymph node metastasis were present in 5/5, 3/5 and 0/5, respectively. Undetectable pentagastrin-stimulated CT levels were achieved and maintained postoperatively in all five children (average follow-up 3.6 years)., Conclusions: MEN 2 guidelines on thyroidectomy can be efficiently and safely implemented by a multidisciplinary team operating in a single centre. The lack of guidelines on cervical neck dissection remains a problem; this could be solved by determining an age under which this procedure would be deemed unnecessary.

Objective: To report on our experience with intraamniotic injection of ethacridine lactate (EL) for second trimester termination of pregnancy (TOP) and to compare its effectiveness with vaginal PGE1 in this setting., Study Design: (a) Retrospective analysis of 54 intraamniotic EL-induced TOP. (b) Matched pair analysis of 50 second trimester TOP, using intraamniotic EL in group A (n=25) and vaginal PGE1 suppositories in group B (n=25)., Main Outcome Measures: success rate, induction-delivery interval, analgesic use, drop in maternal hemoglobin, complications., Results: (a) TOP with EL alone was successful in 50 cases (93%). Mean duration from instillation to delivery was 40.5 h (range 14-58, S.D.: +/-9.0). Mean analgesic need was low. Mean drop in hemoglobin was low (1.0 g/dl, S.D.: +/-1.1). (b) The matched pair analysis revealed a significantly shorter induction-delivery interval in the vaginal PGE1 group (mean 26.9 h, range 8-80, S.D.: +/-16.8 versus 41.0 h, range 24-55, S.D.: +/-7.3, p<.05). There were no significant differences in analgesic use or drop in hemoglobin. In both groups one case of post partum hemorrhage occurred., Conclusion: Intraamniotic instillation of ethacridine lactate in second trimester TOP is safe, cheap and successful in the vast majority of cases. Disadvantages are the longer induction-delivery interval compared to modern prostaglandins and the invasive mode of application. However, by the use of EL the side effects of repeated prostaglandin application can be avoided as well as multiple vaginal examinations. Furthermore, the time of delivery can be predicted in a narrower interval.

Aberrant expression of ectopic G protein-coupled receptors (GPCRs) in adrenal cortex tissue has been observed in several cases of ACTH-independent macronodular adrenal hyperplasias and adenomas associated with Cushing's syndrome. Although there is clear clinical evidence for the implication of these ectopic receptors in abnormal regulation of cortisol production, whether this aberrant GPCR expression is the cause or the consequence of the development of an adrenal hyperplasia is still an open question. To answer it, we genetically engineered primary bovine adrenocortical cells to have them express the gastric inhibitory polypeptide receptor. After transplantation of these modified cells under the renal capsule of adrenalectomized immunodeficient mice, tissues formed had their functional and histological characteristics analyzed. We observed the formation of an enlarged and hyperproliferative adenomatous adrenocortical tissue that secreted cortisol in a gastric inhibitory polypeptide-dependent manner and induced a mild Cushing's syndrome with hyperglycemia. Moreover, we show that tumor development was ACTH independent. Thus, a single genetic event, inappropriate expression of a nonmutated GPCR gene, is sufficient to initiate the complete phenotypic alterations that ultimately lead to the formation of a benign adrenocortical tumor.

Hepatocellular carcinoma (HCC) with lymphocytic infiltration is a rare entity recently described, sometimes associated with hepatitis C. Histologically, remarkable inflammatory cell infiltration of cancer nests is observed, mostly composed of T cytotoxic lymphocytes. When prominent, this inflammatory cell component can obscure the neoplastic cells, leading to diagnostic difficulty. Alike tumors showing dense lymphocytic infiltrate, it discloses a better prognosis than other HCC. We report a case of HCC with lymphocytic infiltration arising in the right suprarenal space, in a 45-year-old man with no chronic liver disease. The patient is alive without recurrence three years after surgical resection. This report is original because HCC growing ectopically are rare and need to be distinguished, specially in the right retroperitoneal, from metastatic adrenal HCC.

Authors report on a case of one intramuscular metastasis from a non-medullary differentiated thyroid carcinoma. Muscular metastasis are rare in this pathology. Moreover, body-scanning scintigraphic interpretation is difficult for the pelvic area. False-positive are numerous and a very precise topographical diagnosis is required to choose the best surgical approach. Authors propose a specific management of different imaging methods for such a metastatic site. They associate a CT scan and a multiplanar iodine and technetium scintigraphy.

Objectives: Histological examination of liver biopsy is currently required in the management of patients with chronic hepatitis C. Our aim was to evaluate the diagnostic utility of a panel of circulating markers in detecting the stage of fibrosis., Methods: One hundred and ninety four-patients who had undergone a percutaneous liver biopsy before antiviral treatment, and 194 age- and sex-matched healthy subjects were studied. Serum levels of hyaluronate, procollagen type III N-terminal peptide (PIIINP), matrix metalloproteinases (MMP)-1, MMP-2, MMP-9 and their tissue inhibitors of metalloproteinases (TIMP)-1 and TIMP-2 were determined by RIA and ELISA. Histological lesions were staged according to the METAVIR score., Results: Hyaluronate, PIIINP, TIMP-1, and TIMP-2 serum levels were significantly higher in patients than in controls. Six markers were significantly correlated with fibrosis: MMP-2 (r = 0.28; p < 0.01), TIMP-1 (r = 0.42; p < 0.001), HA (r = 0.50; p < 0.001), PIIINP (r = 0.62; p < 0.0001), MMP-1 (r = -0.32; p < 0.01), and MMP-9 (r = -0.22; p < 0.05). By multivariate analysis, only PIIINP and MMP-1 were independently associated with fibrosis, and were combined using the equation of the logistic regression model. Using receiver-operating characteristics analysis, the area under the curve of the score to discriminate mild (FO/F1) from significant fibrosis (F2/F3/F4) was 0.82, with a sensitivity of 60% for a specificity of 92%., Conclusion: Our results suggest that combining two serum markers reflecting fibrogenesis (PIIINP) and fibrolysis (MMP-1) may provide a useful tool for evaluating liver fibrosis.

Trypanosomes isolated from South American bats include the human pathogen Trypanosoma cruzi. Other Trypanosoma spp. that have been found exclusively in bats are not well characterized at the DNA sequence level and we have therefore used the SL RNA gene to differentiate and characterize kinetoplastids isolated from bats in South America. A Trypanosoma sp. isolated from hats in southern Brazil was compared with the geographically diverse isolates T. cruzi marinkellei, T. vespertilionis, and T. dionisii. Analysis of the SL RNA gene repeats revealed size and sequence variability among these bat trypanosomes. We have developed hybridization probes to separate these bat isolates and have analysed the DNA sequence data to estimate their relatedness. A new species, Trypanosoma desterrensis sp. n., is proposed, for which a 5S rRNA gene was also found within the SL RNA repeat.

Purpose: Bronchial carcinogenesis is a multistep process characterized by accumulation of genetic and molecular abnormalities, which precedes and accompanies the preinvasive lesions known as dysplasia and carcinoma in situ (CIS). We hypothesized that the level of accumulated molecular abnormalities in dysplasia assessed by immunohistochemical markers might reflect the severity of the carcinogenic process, thus allowing for risk assessment in smokers., Experimental Design: We performed a prospective analysis of bronchial biopsies in 48 former smokers who had at least one area of metaplasia. Twenty-two of the patients had a previous history of lung cancer. Eighty bronchial lesions were recorded at baseline, including 31 metaplasia, 12 mild dysplasia, 9 moderate dysplasia, 9 severe dysplasia, and 19 CISs. Forty-one percent of the patients had multiple preinvasive lesions. Immunohistochemical analysis of P53, cyclin D1, cyclin E, Bax, and Bcl2 was performed. Aberrant expression of one of these proteins as compared with normal bronchi was recorded as one molecular alteration., Results: After 18 months, 17 patients were diagnosed with lung cancer. No isolated parameter, including dysplastic grade or any isolated molecular alteration, was significantly associated with cancer occurrence at 18 months follow-up, using a logistic regression statistical analysis. In contrast, considering CIS and cancer as end point, more than two immunohistochemical abnormalities were associated with cancer or CIS occurrence (P = 0.02)., Conclusions: We concluded that the cumulative index of immunohistochemical abnormalities in a random dysplasia is associated with CIS or lung cancer in the cancerization field of symptomatic smokers, independently of the histopathological grade of dysplasia. This set of histopathological biomarkers might be useful in risk assessment and provide intermediate end points for chemopreventive trials.

The international standard for histologic classification of lung tumours is that proposed by the WHO/IASLC (1999). Ninety percent of these cancers are distributed in squamous cell carcinoma, adenocarcinoma, small cell carcinoma, and large cell carcinoma, all related to tobacco smoke carcinogens, responsible for 90% of tumours. As a consequence of their derivation from totipotential stem cells, heterogeneity is common. They arise from multifocal preinvasive lesions reflecting the "field cancerization" process of the entire bronchial tree. All together 1999 WHO classification offers a valuable hallmark for clinical, biological, and epidemiological studies, and a solid basis for inclusion of patients in randomized therapeutical trials as well as for interpretation of sensibility or resistance to new therapeutic agents.

We collected 75 primary pulmonary carcinomas with pleomorphic, sarcomatoid, or sarcomatous elements to better define their clinical, histologic, and immunohistochemical profile. The patient's age ranged from 42 to 81 years (mean 65 years), and the male-to-female ratio was 9.7:1. Sixty-nine patients (92%) were smokers. Cough and hemoptysis were the most frequent presenting symptoms. Fifty-nine patients (65%) died of disease: only stage significantly predicts overall survival (p = 0.0273). Microscopically, based on the WHO criteria, 58 cases were classified as pleomorphic carcinoma (51 with an epithelial component, 7 composed exclusively of spindle and giant cells), 10 as spindle cell carcinoma, 3 as giant cell carcinoma, 3 as carcinosarcoma, and 1 as pulmonary blastoma. Immunohistochemically, in the tumors composed exclusively of spindle and/or giant cells, thyroid transcription factor-1 (TTF-1) and cytokeratin 7 were positive in 55% and 70% of the cases, respectively, whereas surfactant protein-A was always negative. In pleomorphic carcinomas with an epithelial component, cytokeratin 7, TTF-1, and surfactant protein-A were positive in the sarcomatoid component in 62.7%, 43.1%, and 5.9% of the cases, respectively, whereas they were always negative in the sarcomatous part of carcinosarcomas and blastoma. In the epithelial component of pleomorphic carcinomas, cytokeratin 7, TTF-1, and surfactant protein-A were positive in 76.4%, 58.8%, and 39.2% of the cases, respectively, whereas the same antibodies did not react with the epithelial component of carcinosarcomas; in the case of blastoma, the epithelial part of the tumor was positive for cytokeratin 7 and TTF-1, whereas it was negative for surfactant protein-A. Cytokeratin 20 was always negative. In our opinion, this study: 1) supports the metaplastic histogenetic theory for this group of tumors; 2) shows that cytokeratin 7 and TTF-1, but not surfactant protein-A, are useful immunohistochemical markers in this setting; 3) confirms that stage is at the moment the only significant prognostic parameter, as in conventional non-small cell lung carcinomas; and 4) shows that this group of tumors has a worse prognosis than conventional non-small cell lung carcinoma at surgically curable stages I, justifying their segregation as an independent histologic type in the WHO classification.

Background/aims: The interval at which liver biopsy should be repeated in untreated patients with chronic hepatitis C is not defined. We examined fibrosis change by METAVIR scoring in these patients in whom two or more liver biopsies were available., Methods: One hundred and eighty patients with histologically proven chronic hepatitis C were studied. Mean delay between biopsies was 3.67+/-2.69 years and 3.08+/-1.43 in the 16 patients having three biopsies. Univariate and multivariate analyses were performed to determine factors associated with liver fibrosis progression., Results: Median rate of fibrosis progression per year was 0.04 (0.00-0.55) to first biopsy, 0.00 (-0.84-1.02) between first and second biopsy (NS), and 0.17 (0.00-1.50) between second and third biopsy (P<0.05). In multivariate analysis, only age at first biopsy >40 years (OR=5) (2-12) and alcohol consumption of 1-50 g per day (OR=4) (2-12) and more than 50 g per day (OR=8) (3-23) were associated with severe fibrosis. The number of patients who increased in fibrosis stage was significantly higher after 4 years (P<0.02)., Conclusions: An interval of at least 4-5 years is needed between liver biopsies to measure change in patients with mild liver disease.

Aims: Monoclonal antibody 34betaE12 (Ck34betaE12) recognizes a set of cytokeratins (1, 5, 10, 14) expressed in normal stratified squamous epithelium. We have recently reported its expression in squamous cell carcinoma and basaloid carcinoma, in contrast to large cell neuroendocrine carcinoma, an entity with overlapping morphological features with basaloid carcinoma. We have now examined the role of Ck34betaE12 in discriminating between neuroendocrine and non-neuroendocrine proliferations., Methods and Results: We performed an immunohistochemical study of 228 cases, comprising the whole spectrum of lung neuroendocrine proliferations and tumours. All cases of neuroendocrine cell hyperplasia (n = 15), tumorlet (n = 23), typical carcinoid (n = 27) and atypical carcinoid (n = 23) were completely negative for Ck34betaE12. Although the neuroendocrine cells of small cell lung carcinoma and large cell neuroendocrine carcinoma were consistently negative, a strong and diffuse positive staining was found in the non-neuroendocrine components of combined small cell carcinoma (three of eight cases) and combined large cell neuroendocrine carcinoma (11 of 12 cases). In addition, scattered Ck34betaE12+ cells were noted in 11 of 64 (17%) large cell neuroendocrine carcinoma and in seven of 56 (12.5%) small cell carcinoma, which were not obviously histologically combined. This heterogeneity of high-grade neuroendocrine tumours was not observed in carcinoids which lack Ck34betaE12 clusters of reactive cells. There was mutual exclusion between expression of neuroendocrine markers and that of Ck34betaE12., Conclusion: We conclude that 34betaE12 expression excludes the neuroendocrine nature of tumour cells and uncovers the real frequency of combined forms in high-grade neuroendocrine tumours.

Bilateral inferior petrosal sinus sampling (BIPSS) is the most reliable procedure for distinguishing Cushing's disease from ectopic ACTH secretion. However, it is less reliable at predicting the lateralization of the pituitary corticotroph microadenoma. We sought to determine whether this could be improved by taking into account the pattern of venous drainage and the precise location of the catheters. We retrospectively studied data from 86 patients who underwent BIPSS. Cushing's disease was predicted in 74 patients, of whom 69 underwent transsphenoidal surgery. Surgical cure was obtained in 65 patients, with identification of a corticotroph microadenoma in 58 cases. In 49 patients the location of the microadenoma predicted by the intersinus ACTH gradient could be compared with the pathologist's data. BIPSS accurately predicted the lateralization of the microadenoma in only 57% of these patients. Prediction was improved to 71% when both venograms and catheters were symmetric (35 patients). In this subgroup accuracy was 86% in patients with both catheters in the inferior petrosal sinuses compared with 50% in patients with both catheters in the cavernous sinuses (CS). Two transient sixth nerve palsies occurred during CS catheterization. Our data suggest that BIPSS results are much improved when venous drainage is symmetric. Catheterization of CS did not improve the results and was less safe.

There is a high level of genetic diversity among the clinical isolates of the taxon Trypanosoma cruzi. The results of recent studies on well defined gene loci have revealed that T. cruzi can be divided into two major phylogenetic lineages, designated T. cruzi I and T. cruzi II. Further intra-lineage polymorphisms were explored, in clinical isolates from five representative regions of Brazil, using the internal transcribed spacers that flank the 5.8S ribosomal RNA gene. These regions were amplified in a PCR and the products were digested with six restriction enzymes before being subjected to RFLP analysis in polyacrylamide gels. The results were used to construct a complex evolutionary tree, in which the isolates were clustered into two major phylogenetic lineages with a low level of similarity. To investigate the evolutionary relationships between the isolates, the PCR products were cloned and sequenced. The results of the subsequent phylogenetic analysis confirmed the presence of the two major groups of T. cruzi and showed that zymodeme 3, a third iso-enzymatic sub-group, is closer to T. cruzi I than to T. cruzi II.

This study evaluated the incidence of atrial premature beats (APBs) and the impact of various provocative maneuvers in patients with focally initiated AF. Fifty patients (39 men, 57 +/- 0.4 years) with focally initiated, paroxysmal AF underwent Holter recording and a standardized protocol of provocative maneuvers: vagomechanical stimulation, adenosine 12 mg i.v., esmolol 500 ng/kg i.v., orciprenaline i.v. 5 mg/50 mL saline 0.9%, and atropine 0.01 mg/kg i.v. A surface ECG was recorded for 20 minutes at baseline and following each part of the protocol. High focal activity was defined as > or = 1 APB/minute. During Holter ECG, 29 (58%) patients had an amount of < 200 APBs, 12 (24%) patients < 700 > or = 200, and 9 (18%) patients > or = 700 APBs. Less than 1 hour of high focal activity was observed in 34 (68%) of the 50 patients. Before starting provocative maneuvers 15 (30%) patients had high focal activity whereas 35 (70%) presented < 1 APB/minute. In 29 (58%) patients APBs increased by > or = 1/min during provocative maneuvers: by vagomechanical stimulation in 11 patients, after adenosine in 15, esmolol in 12, orciprenaline in 15, and after atropine in 9 patients. In all patients with > or = 1 APB/min at baseline, focal activity decreased or disappeared during some single provocative maneuvers. AF occurred in eight patients under provocative maneuvers. No predictive factors of a successful provocative maneuver were detected with regard to the baseline patients characteristics and Holter results. In conclusion, patients with focally initiated AF have a low incidence of spontaneous APBs. Various provocative maneuvers successfully increase APBs in more than half of the patients; orciprenaline had the highest efficacy. Some provocative maneuvers may suppress APBs in the setting of high focal activity at baseline.

The World Health Organization's classification of lung tumors separately categorizes neuroendocrine (NE) lung tumors, small cell lung carcinoma (SCLC), and large cell neuroendocrine carcinoma (LCNEC) as high-grade NE malignancies and carcinoids (typical, [TC] and atypical [AC]) as low- and intermediate-grade malignancies. Although these NE tumors are considered with NE hyperplasia (NEH) and tumorlets as part of a spectrum of NE proliferations, their derivation from a common progenitor cell has not received full agreement. With the aim of refining their differential diagnosis and extending our understanding of their histogenesis, we studied the expression of thyroid transcription factor-1 (TTF-1), a transcription factor that regulates lung morphogenesis and differentiation, along the spectrum of NE lung tumors. Two hundred and twenty- seven NE proliferations and tumors were immunostained with TTF-1 antibody. Positive immunostaining for TTF-1 was detected in 47 of 55 (85.5%) pure SCLCs, in 31 of 64 (49%) pure LCNECs, but in none of 15 NEHs, 23 tumorlets, or 50 carcinoid tumors (27 TCs and 23 ACs). In 19 of 20 (95%) combined SCLCs and LCNECs, TTF-1 expression was identical in both NE and non-NE components. These results show that TTF-1 is not expressed in normal and hyperplastic NE cells or in carcinoids, but is expressed in high-grade NE proliferations and in lung adenocarcinomas. This challenges the concept of a spectrum of NE proliferations and tumors and lends credence to the alternative hypothesis of a common derivation for SCLC and non-SCLC including LCNEC, with carcinoids deriving from a different stem cell., (Copyright 2002, Elsevier Science (USA). All rights reserved.)

We report the cloning and sequencing of the first minicircle from a phloem-restricted, pathogenic Phytomonas sp. (Hart 1) isolated from a coconut palm with hartrot disease. The minicircle possessed a two-domain structure of two conserved regions, each containing three conserved sequence blocks (CSB). Based on the sequence around CSB 3 from Hart 1, PCR primers were designed to allow specific amplification of Phytomonas minicircles. This primer pair demonstrated specificity for at least six groups of plant trypanosomatids and did not amplify from insect trypanosomatids. The PCR results were consistent with a two-domain structure for other plant trypanosomatids.

The spliced leader RNA genes of Bodo saltans, Cryptobia helicis and Dimastigella trypaniformis were analyzed as molecular markers for additional taxa within the suborder Bodonina. The non-transcribed spacer regions were distinctive for each organism, and 5S rRNA genes were present in Bodo and Dimastigella but not in C. helicis. Two sequence classes of 5S rRNA were evident from analysis of the bodonid genes. The two classes of 5S rRNA genes were found in other Kinetoplastids independent of co-localization with the spliced leader RNA gene.

Basaloid carcinoma (BC) and large-cell neuroendocrine carcinoma (LCNEC) are 2 recently recognized variants of large-cell lung carcinomas that may overlap in their morphology, and are discriminated by expression of neuroendocrine markers in LCNEC. Because thyroid transcription factor 1 (TTF-1) is expressed in lung adenocarcinomas but not in squamous cell carcinomas (SCC), and 34betaE12 recognizes a set of high-molecular-weight cytokeratins characteristic of basal stem cells, we hypothesized that these 2 markers could help in distinguishing BC from LCNEC. Immunostaining for TTF-1 was detected in 40.9% of pure LCNEC but in no BC or basaloid variant of SCC. In contrast, immunoreactivity for 34betaE12 was shown in all BC and basaloid variant of SCC but in only 1 LCNEC. Bouin fixation was less efficient than formalin in the immunodetection of both markers for its well-known deleterious effect on antigen preservation. Specificity of TTF-1 for LCNEC (100%) and that of 34betaE12 for BC (98.3%) exceeded that of NE markers for distinction of these 2 entities. These data show that TTF-1 and 34betaE12, in association with specific neuroendocrine markers, represent a useful panel of antibodies in differentiating carcinomas presenting with a solid pattern, palisading, or pseudorosettes, the expression of TTF-1 excluding the diagnosis of BC, and staining with 34betaE12 excluding pure LCNEC., (Copyright 2001 by W.B. Saunders Company)

The phylogenetic placement of the genus Diplonema in relation to fellow phylum members Euglena and Trypanosoma has been uncertain. The spliced leader RNA gene, present in the euglenids and kinetoplastids in distinct forms, was a potential target for resolving this question. The first indication supporting a closer relationship to the kinetoplastids was the recognition of potential spliced leader RNA exon sequences in the genomic DNA of two Diplonema isolates. Examination of total cell RNA revealed transcripts in the anticipated size range at approximately 120 and 130 nt. Specific PCR amplification of a spliced leader RNA gene repeat was performed. The hallmark features of the kinetoplastid-type spliced leader RNA, specifically the 39-nt exon, splice-donor site, Sm-binding site and poly-T tract and the potential to form the requisite stem-loop structures, were found. Diplonema spp. are different from the kinetoplastids by virtue of C residues at positions 4 and 18 in the exon. While the intergenic spacer regions varied in size, each contained the complete sequence or remnants of a 5S ribosomal RNA gene. Possession of a functional spliced leader RNA gene of the kinetoplastid variety in Diplonema supports a closer evolutionary relationship with this group than with the euglenids.

The arbitrary genus Phytomonas includes a biologically diverse group of kinetoplastids that live in a wide variety of plant environments. To understand better the subdivisions within the phytomonads and the variability within groups, the exon, intron and non-transcribed spacer sequences of the spliced leader RNA gene were compared among isolates of the phloem-restricted members. A total of 29 isolates associated with disease in coconut, oil palm and red ginger (Alpinia purpurata, Zingibreaceae) were examined, all originating from plantations in South America and the Caribbean over a 12-year period. Analysis of non-transcribed spacer sequences revealed 2 main groups, I and II; group II could be further subdivided into 2 subgroups, IIa and Ilb. Three classes of spliced leader (SL) RNA gene were seen, with SLI corresponding to group I, SLIIa to group lIa, and SLIIb to group IIb. Two isolates showed some characteristics of both major groups. Group-specific oligonucleotide probes for hybridization studies were tested, and a multiplex amplification scheme was devised to allow direct differentiation between the 2 major groups of phloem-restricted Phytomonas. These results provide tools for diagnostic and molecular epidemiology of plant trypanosomes that are pathogenic for commercially important flowers and palms.

To elucidate the process of transcription in the kinetoplastids and to aid in the purification of transcription factors, we have developed a transcriptionally-competent nuclear extract from Leishmania tarentolae for the study of the spliced leader (SL) RNA gene. The extract was competent to transcribe a tagged SL RNA gene. The in vitro SL RNA transcripts initiated accurately and their synthesis was dependent on the presence of the promoter defined in vivo. The nuclear extract was then challenged rigorously using an exhaustive set of mutated SL RNA gene templates previously tested for transcriptional activity in vivo. Mutation of four nucleotides (CCGG) at positions -34 to -31 had a detrimental effect on transcription in vitro: the CC dinucleotide overlaps one element necessary in vivo. Similarly. four nucleotides (TGAC; positions -67 to -64) important for transcription in vitro overlapped the other core promoter element defined in vivo, but were generally not effective as point mutations. The promoter-binding ability of the transcriptionally-competent extract for the -60 region mutations mirrored the in vitro transcription pattern. Although it does not reflect precisely the in vivo results, this in vitro system provides us with an important tool for monitoring the purification of potential transcription factors, as well as the basis for future reconstitution experiments.

Trypanosomatids isolated from plants have been assigned typically into the genus Phytomonas. Such designations do not reflect the biology of the diverse isolates; confusion may arise due to the transient presence in plants of monogenetic (insect) trypanosomatids deposited by phytophagous bugs. To develop further molecular markers for the plant kinetoplastids, we have obtained the DNA sequence of the 5S ribosomal RNA gene from 24 isolates harvested from phloem, latex, and fruit. Small, distinct sequence differences were found at the 3'-ends of the transcribed regions; substantial sequence and size differences were found in the non-transcribed regions. Alignment of the gene sequences from all the isolates suggested the presence of eight groupings. While six groups contained isolates from single plant tissues, groups C and A contained isolates from both fruit and latex. The DNA sequences of the 10 phloem-restricted pathogenic isolates from South America and the Carribean were highly conserved and thus comprised a single group (H). The conserved nature of the 5S ribosomal RNA genes in these plant pathogens supports the proposal that they be considered as a distinct section, the phloemicola.

The classification of neuroendocrine (NE) lung tumors has been revised in the 1999 World Health Organization (WHO) classification of lung tumors, allowing sharp morphological definition of typical versus atypical carcinoids, and atypical carcinoids versus large cell neuroendocrine carcinoma (LCNEC), a newly described class of high-grade NE lung tumors which differs from small-cell lung cancer by a large-cell phenotype. Divergent differentiation accounts for the high frequency of glandular differentiation with mucin production, and ultrastructural features in carcinoids and LCNEC, and low frequency of squamous differentiation in both LCNEC and SCLC. Specific NE markers (chromogranin, synaptophysin, neural cell adhesion molecule) and epithelial markers consistently negative in neuroendocrine components (cytokeratins 1, 5, 10, 14; epidermal growth factor (EGF)-receptor, human leukocyte antigen beta 2 (HLA-beta2) microglobuline) help to recognize divergent differentiation in NE tumors. At morphological level, divergent differentiation in NE tumors is recognized in WHO classification as variants: combined SCLC and combined LCNEC. The derivation of all lung tumors from a common endodermal stem cell and adoption of amine precursor uptake and decarboxylation properties by this endodermal stem cell explains divergent differentiation in NE lung tumors and the occurrence of NE subsets in NSCLC.

Neural cell adhesion molecules (NCAM) are adhesion molecules expressed by neural and neuroendocrine tumors and a few biphasic tumors such as synovialosarcomas and breast phyllode tumors. To investigate NCAM expression in mesotheliomas, we studied 26 cases of epithelioid (n = 12), biphasic (n = 11), and sarcomatoid (n = 3) malignant mesotheliomas (MM), in comparison with normal mesothelium, and 50 primary non-small cell lung carcinomas (NSCLC) (25 adenocarcinomas [ADC] and 25 squamous cell carcinomas [SCC]), using electron microscopy as a gold standard for recognition of MM. NCAM reactivity using 123C3 antibody was compared with that of NE markers such as chromogranin A and synaptophysin. Although normal mesothelium remains negative, NCAM was expressed in 19 of 26 MM (73%) with a membranous staining on frozen or paraffin sections. In 6 of 12 epithelioid MM, the tumor cells expressed NCAM, whereas in 5 cases stromal fibroblasts showed a strong but focal staining. In 11 biphasic MM, 4 presented an NCAM reactivity of both epithelioid and spindle cell components, whereas in 7, only fusiform component was NCAM positive. Two of 3 sarcomatoid MM showed an NCAM expression. Chromogranin expression was never seen, whereas synaptophysin was noticed in 2 cases. No case of NSCLC showed membranous 123C3 staining, whereas 2 ADC weakly expressed synaptophysin. We conclude that NCAM expression in MM is reminiscent of its expression in mesoderm during fetal life and consistent with that reported in other biphasic tumors. These data show that NCAM expression occurs in 73% of MM, highly exceeding that observed in lung cancer.

In recent years, much has been learned about the cis-elements controlling transcription of the kinetoplastid spliced leader (SL) RNA gene. The SL RNA gene contains the first 39 nucleotides that are trans-spliced on to all nuclear-derived mRNAs in these organisms. Transcription initiation is determined by two precisely spaced upstream elements and transcription termination is directed by the downstream poly-T tract, although the RNA polymerase responsible for SL RNA synthesis is still questioned. In this article, David Campbell, Nancy Sturm and Michael Yu review the field of kinetoplastid SL RNA gene transcription, address past proposals in light of current data and discuss some of the differences that appear in the literature.

A 39-nucleotide leader is trans-spliced onto all trypanosome nuclear mRNAs. The precursor spliced leader RNA was tested for trans-splicing function in vivo by mutating the intron. We report that in Leishmania tarentolae spliced leader RNA 5' modification is influenced by the primary sequence of stem-loop II, the Sm-binding site, and the secondary structure of stem-loop III. The sequence of stem-loop II was found to be important for cap 4 formation and splicing. As in Ascaris, mutagenesis of the bulge nucleotide in stem-loop II was detrimental to trans-splicing. Because restoration of the L. tarentolae stem-loop II structure was not sufficient to restore splicing, this result contrasts the findings in the kinetoplastid Leptomonas, where mutations that restored stem-loop II structure supported splicing. Methylation of the cap 4 structure and splicing was also dependent on both the Sm-binding site and the structure of stem-loop III and was inhibited by incomplete 3' end processing. The critical nature of the L. tarentolae Sm-binding site is consistent with its essential role in the Ascaris spliced leader RNA, whereas in Leptomonas mutation of the Sm-binding site and deletion of stem-loop III did not affect trans-splicing. A pathway for Leishmania spliced leader RNA processing and maturation is proposed.

Addition of a 39-nucleotide (nt) spliced leader (SL) by trans splicing is a basic requirement for all trypanosome nuclear mRNAs. The SL RNA in Leishmania tarentolae is a 96-nt precursor transcript synthesized by a polymerase that resembles polymerase II most closely. To analyze SL RNA genesis, we mutated SL RNA intron structures and sequence elements: stem-loops II and III, the Sm-binding site, and the downstream T tract. Using an exon-tagged SL RNA gene, we examined the phenotypes produced by a second-site 10-bp linker scan mutagenic series and directed mutagenesis. Here we report that transcription is terminated by the T tract, which is common to the 3' end of all kinetoplastid SL RNA genes, and that more than six T's are required for efficient termination in vivo. We describe mutants whose SL RNAs end in the T tract or appear to lack efficient termination but can generate wild-type 3' ends. Transcriptionally active nuclear extracts show staggered products in the T tract, directed by eight or more T's. The in vivo and in vitro data suggest that SL RNA transcription termination is staggered in the T tract and is followed by nucleolytic processing to generate the mature 3' end. We show that the Sm-binding site and stem-loop III structures are necessary for correct 3'-end formation. Thus, we have defined the transcription termination element for the SL RNA gene. The termination mechanism differs from that of vertebrate small nuclear RNA genes and the SL RNA homologue in Ascaris.

We report the cloning and characterization of a gene, LtARL, which encodes a small GTP-binding protein, from the protozoan Leishmania tarentolae. Hybridization analysis of genomic DNA under high stringency conditions indicates the single-copy nature of LtARL. LtARL is transcribed and yields a approximately 0.9 kb mRNA that is processed at the 5' end by trans-splicing. When expressed in Escherichia coli, LtArl binds GTP with a low stoichiometry and in a phospholipid-independent manner. Based on the greatest sequence identity with Homo sapiens Arl3 and lipid-independent binding of guanine nucleotides we designate this gene LtARL and the encoded protein LtArl.

The mini-exon gene repeats from two isolates of Trypanosoma (Nannomonas) simiae were amplified by polymerase chain reaction (PCR). The products from each isolate differed in size, however they cross-hybridised in Southern blots. The nature of the size variation was revealed by comparison of the DNA sequence of each repeat: relative to the BAN7 strain, the mini-exon gene of KETRI 2431 contained three apparent deletions that were flanked by short (> or = 6-bp) direct repeats. Furthermore, one of the cloned repeats was used as a hybridisation probe against DNA from other closely-related African trypanosomes. The lack of hybridisation of the T. (N.) simiae mini-exon gene to genomic DNA from the Forest, Kilifi and Savannah subgroups of T. (N.) congolense and T. (N.) godfreyi indicate that this PCR-hybridisation assay may be useful for distinguishing T. (N.) simiae from other members of the subgenus Nannomonas.

In Kinetoplastid protozoa, trans-splicing is a central step in the maturation of nuclear mRNAs. In Leishmania, a common 39 nt spliced-leader (SL) is transferred via trans-splicing from the precursor 96 nt SL RNA to the 5' terminus of all known protein-encoding RNAs. In this study, promoter elements of the L. tarentolae SL RNA gene have been identified with respect to transcriptional activity and putative transcription factor binding. We have mapped the essential regions in the SL RNA gene promoter at single nucleotide resolution using both in vivo transcription and in vitro protein/DNA binding approaches. Two regions located upstream of the SL RNA gene were identified: a GN3CCC element at -39 to -33 and a GACN5G element at -66 to -58 were essential for SL RNA gene transcription in stably transfected cells. Consistent with other known bipartite promoter elements, the spacing between the GN3CCC and GACN5G elements was found to be critical for proper promoter function and correct transcription start point selection, as was the distance between the two elements and the wild-type transcription start point. The GACN5G element interacts specifically and in a double-stranded form with a protein(s) in Leishmania nuclear extracts. The degree of this protein DNA interaction in vitro correlated with SL RNA gene transcription efficiency in vivo, consistent with a role of the protein as a transcription factor. The core nucleotides GACN5G fit the consensus PSE promoter structure of pol II-transcribed snRNA genes in metazoa.

First characterized in Trypanosoma brucei, the spliced leader-associated (SLA) RNA gene locus has now been isolated from the kinetoplastids Leishmania tarentolae and Trypanosoma cruzi. In addition to the T. brucei SLA RNA, both L. tarentolae and T. cruzi SLA RNA repeat units also yield RNAs of 75 or 76 nucleotides (nt), 92 or 94 nt, and approximately 450 or approximately 350 nt, respectively, each with significant sequence identity to transcripts previously described from the T. brucei SLA RNA locus. Cell fractionation studies localize the three additional RNAs to the nucleolus; the presence of box C/D-like elements in two of the transcripts suggests that they are members of a class of small nucleolar RNAs (snoRNAs) that guide modification and cleavage of rRNAs. Candidate rRNA-snoRNA interactions can be found for one domain in each of the C/D element-containing RNAs. The putative target site for the 75/76-nt RNA is a highly conserved portion of the small subunit rRNA that contains 2'-O-ribose methylation at a conserved position (Gm1830) in L. tarentolae and in vertebrates. The 92/94-nt RNA has the potential to form base pairs near a conserved methylation site in the large subunit rRNA, which corresponds to position Gm4141 of small rRNA 2 in T. brucei. These data suggest that trypanosomatids do not obey the general 5-bp rule for snoRNA-mediated methylation.

Every kinetoplastid mRNA receives a common, conserved leader sequence via the process of trans-splicing. In Leishmania tarentolae the precursor spliced leader RNA is 96 nucleotides, with a 39-nucleotide exon that is 7meG-capped and methylated on the first 4 nucleotides. trans-Splicing was inferred from the presence of tagged leader in the high molecular weight RNA population and confirmed for accuracy by cDNA cloning. Linker scan substitutions within the exon between positions 10 and 39 did not affect trans-splicing. The trans-splicing efficiency for three of the scan exons was proportional to the tagged:wild type ratio in the spliced leader precursor population. Two scan leader RNAs that were efficiently spliced showed reduced methylation. Longer exons showed reduced splicing, whereas 10- or 20-base pair deletions abolished splicing. These results indicate that size, but not content, of the exon is a constraint on the splicing process. These results, in combination with previous data eliminating a role in transcription initiation, suggest that translation may be the selective pressure on the leader content.

We have designed and synthesized eight compounds 2-9 which incorporate various amino acid residues in positions 17, 18, and 21 of the glucagon molecule: 2, [Lys17]glucagon amide; 3, [Lys18]glucagon amide; 4, [Nle17,Lys18,Glu21]glucagon amide; 5, [Orn17,18, Glu21]glucagon amide; 6, [d-Arg17]glucagon; 7, [d-Arg18]glucagon; 8, [d-Phe17]glucagon; and 9, [d-Phe18]glucagon. Compared to glucagon (IC50 = 1.5 nM), analogues 2-9 were found to have binding affinity IC50 values (in nM) of 0.7, 4.1, 1.0, 2.0, 5.0, 25.0, 43.0, and 32.0, respectively. When these compounds were tested for their ability to stimulate adenylate cyclase (AC) activity, they were found to be full or partial agonists having maximum stimulation values of 100, 100, 100, 100, 87, 78, 94, and 100%, respectively. On the basis of the X-ray crystal structure of [Lys17,18,Glu21]glucagon amide reported here, the ability to form a salt bridge between Lys18 and Glu21 is probably key to their increased binding and second messenger activities. Among the eight analogues synthesized here, only analogue 4 preserves the ability to form a salt bridge between Lys18 and Glu21. However, since these modifications are minor they do not seem to change the amphiphilic character of the C-terminus, allowing these analogues to reach 78-100% stimulation in the adenylate cyclase assay. Biological data from analogues 6-9 supports the idea that position 18 of glucagon may influence binding only, while position 17 may influence both receptor recognition and transduction.

To develop molecular markers for lower trypanosmatids, we have examined the mini-exon gene repeats of 17 isolates that were classified as Crithidia by traditional methods. Representative repeats were amplified by polymerase chain reaction and the amplification products were cloned and used as hybridization probes against genomic DNA. Six hybridization groups of Crithidia were defined on the basis of the DNA blotting experiments. The three endosymbiont-bearing species (C. deanei, C. desouzai and C. oncopelti) and C. acanthocephali each belonged to single-member hybridization groups, while the C. fasciculata group contained additional named and undesignated species. The Crithidia lucilae thermophila probe hybridized to multiple undesignated isolates. The DNA sequence of the cloned products revealed that the specificity of the hybridization probes was due to substantial differences in the intron and the nontranscribed spacer regions. These data indicate substantial heterogeneity within the mini-exon gene locus of the taxon Crithidia.