Migraine and tension-type headaches impose a tremendous economic drain upon the healthcare system. Intravenous and oral niacin has been employed in the treatment of acute and chronic migraine and tension-type headaches, but its use has not become part of contemporary medicine, nor have there been randomized controlled trials further assessing this novel treatment. We aimed to systematically review the evidence of using intravenous and/or oral niacin as a treatment for migraine headaches, tension-type headaches, and for headaches of other etiologic types.We searched English and non-English language articles in the following databases: MEDLINE (1966–February 2004), AMED (1995–February 2004) and Alt HealthWatch (1990–February 2004).Nine articles were found to meet the inclusion criteria and were included in this systematic review. Hypothetical reasons for niacin's effectiveness include its vasodilatory properties, and its ability to improve mitochondrial energy metabolism. Important side effects of niacin include flushing, nausea and fainting.Although niacin's mechanisms of action have not been substantiated from controlled clinical trials, this agent may have beneficial effects upon migraine and tension-type headaches. Adequately designed randomized trials are required to determine its clinical implications. Migraines and tension-type headaches impose an important burden upon society and the working public. According to the National Headache Foundation, some 45 million Americans suffer from chronic, recurring headaches and 28 million of these suffer from migraine headaches annually . FurtherEven though advances have been made with regard to the treatment of acute migraine headaches (i.e., the triptan formulations), many patients often discontinue their migraine interventions due to treatment dissatisfaction . Among iOne novel, but not really new treatment option, is the administration of niacin (nicotinic acid) through intravenous and/or oral routes. Niacin is a well-known over-the-counter (OTC) supplement primarily used for its ability to favorably influence cholesterol levels. Recently, there have been anecdotal reports demonstrating the effectiveness of niacin for aborting acute migraine attacks , and forWe conducted a systematic search of English and non-English language articles in the following databases: MEDLINE (1966–February 2004), AMED (1995–February 2004) and Alt HealthWatch (1990–February 2004). Articles were searched with the key search terms "Migraine" combined with the Boolean Operator "AND "Niacin" OR "Nicotinic Acid." Additional searches were conducted with the search terms "Headache" and "Tension." To supplement the search, we searched through the references of the articles we found from the databases.To be included in our final review, articles had to report on the use and administration of niacin for migraine or any other types of headache. We included articles assessing original reports in both peer-reviewed and non-peer-reviewed journals.We determined the evidence grade of each report found, based on the hierarchy of evidence developed by the Oxford Centre for Evidence Based Medicine . Table 1A total of 14 articles were screened ,7,9-20. This case series of 21 patients was limited as it was uncontrolled and involved a small number of patients. The methods that were used to evaluate efficacy of the treatment were primarily based upon a subjective questionnaire or medical record. The results would have been more meaningful if all the patients used the questionnaire to evaluate their treatment responses. Finally, although the symptomatic changes were witnessed immediately following niacin injection, the lifestyle recommendations may have had therapeutic benefits as well.This study involved 100 patients having headaches of multiple etiologies. This study was not properly controlled, but did at least provide some comparison against a group of patients that were not administered the flush forms of intravenous niacin. The fact that the control group did not substantially benefit from the intravenous niacinamide lends more therapeutic efficacy to the ability of intravenous sodium nicotinate or niacin to have a marked therapeutic effect. The sample size was sufficient in number (n = 100), but the determination of therapeutic effectiveness was purely subjective and did not include any questionnaire or standardized method of evaluation.Side effects were minimal; 2 patients experienced mild abdominal cramps, 1 patient vomited but was suspected as having a pathologic condition of the stomach, and 1 other patient with migraine vomited forty-five minutes after the injection. A few patients found the treatment to be worse than their headaches. The authors concluded that the relief of headaches seemed to be correlated with the degree of flushing from the sodium nicotinate or niacin, and that this therapy was most useful among the migraine, spinal tap, and idiopathic groups.A number of shortcomings were evident in this article. The first of which was that the study was not properly controlled and did not contain a placebo group or a group of patients acting as self-controls. However, some of the patients were given intravenous treatments on more than one occasion. This procedure helped in determining treatment reliability and reproducibility since niacin appeared to achieve therapeutic benefits on several occasions. There were no standardized methods of evaluating efficacy since the treatment responses were based upon the medical record and subjective reports. The results would have been more meaningful if all the patients used a standardized questionnaire prior to and after each treatment. In addition, there was only one male subject and 14 females in the study.In this study of 35 patients were given treatments of intravenous dihydroergotamine methanesulfonate, intravenous niacin, or oral combination tablets of ergotamine tartrate and caffeine. Although no control group was used in this study, the patients were given multiple treatments on several occasions. This offered an interesting comparison to be made between niacin and other treatments. Since no control or placebo group was included, it cannot be determined if the therapeutic results were due, in part, to chance or placebo effects. The results might have been more meaningful if all the patients were given a standardized questionnaire prior to and after each treatment, and if an objective measure was incorporated to further substantiate patient responses.These cases reported were well described and clearly demonstrated therapeutic responses during the intravenous and oral niacin therapy. The results would have been more reliable if a comparison had been made to a control group or to a similar patient cohort that were not given the same treatments. Even if the patients served as self-controls, and were told to stop the niacin treatment for a specified period of time, more information could have been gained from their therapeutic responses to niacin. Overall, this report of five cases provides an interesting approach to patients having chronic tension headaches and depression. Its value is limited by the difficulty in extrapolating these findings to a greater number of patients.In this study involving 50 patients there was no data that listed pertinent identifying information, treatment response, past treatments, and duration of treatment for each patient. This would have strengthened the report by being more descriptive, and thus more amenable to critical analysis. If a comparison were made to a control group or a similar patient cohort not given the same treatments, more validity could be have been ascribed to this method of treatment. The patients could have also served as their own controls, thus providing more information about the therapeutic responses to niacin. It cannot be determined if the therapeutic results were due, in part, to chance or placebo. No method of evaluating efficacy was mentioned, except that the responses to niacin were "very satisfactory" in 44 of 50 cases. The results would have been more meaningful if all the patients were given a standardized questionnaire prior to and after each treatment, and if an objective measure was incorporated to substantiate their responses to niacin.In this report, Hall describes the use of niacin for his migraine headaches remarking that the migraines resolved when intense flushing occurred. According to Hall, niacin's benefits and side effects might be due to its ability to release serotonin and histamine from the stomach. There is no reason to doubt Hall when describing his therapeutic response to niacin. However, his report was brief, had no control and was entirely subjective as he was the participant as well as examiner.In this report of 2 cases, it was found that in both cases migraines were relieved with oral niacin. The report would have been more rigorous if the patients had acted as their own self-controls. The value of the report is further diminished by the difficulty in extrapolating these findings to a greater number of patients.This case report was of a 62-year-old woman with a 40-year history of migraine headaches. The patient never acted as her own self-control, which would have made the findings of the case report more meaningful. The fact that the patient's migraine headaches increased in severity after a reduction in dosage does lend more support to niacin as being a migraine preventive agent. The hypothesized increase in serotonin from niacin administration cannot be proven given the limited amount of data contained in the case report. Like all case reports, its value is diminished by the difficulty in extrapolating these findings to a greater number of patients.The results of this systematic review indicate that niacin may have a therapeutic effect on migraine headaches, tension-type headaches, and headaches of other etiologies. The quality of the evidence at this point, however, is only hypothesis generating, and randomized trials are required to determine the clinical implications of this novel treatment.There are several important limitations to consider in the interpretation of this review. We did not find any randomized or controlled trials of niacin on these headaches. We cannot determine to what extent publication bias has on the results of this review. We are unable to draw clinical inferences on the results of the included studies as they were of low quality and have a low level of external generalizability. Despite these limitations, we attempted to conduct an exhaustive search and included all reports of relevance.2 (PGD2) in the skin, leading to a marked increase of its metabolite, 9α, 11β-PGF2, in the plasma [-1 M aqueous methylnicotinate on the forearm, PGD2 is markedly released in the skin and its metabolite appears in high amounts in the plasma [2 causes vasodilation of the intracranial arteries, but niacin's ability to abort acute migraine headaches suggests that this might be what is occurring. Old reports cited by Bicknell and Prescott [Reasons for niacin's effectiveness can only be considered hypothetical, and require clarification from future randomized controlled trials. In acute migraine headaches some of the symptoms arise from activation of the trigeminovascular complex. Activation of this complex leads to intracranial vasoconstriction causing the migraine aura, followed by headache due to vasodilation of the extracranial vessels and activation of the perivascular nociceptive nerves . When tae plasma . When nie plasma ,23. It iPrescott , demonstIn terms of tension-type headaches, it appears that intravenous niacin is of benefit acutely due to its presumed central vasodilatory properties. Like migraines, part of the underlying pathophysiology of chronic tension-type headaches involves central mechanisms, such as the trigeminal system . ChronicSome of the reports did demonstrate prophylactic benefits when niacin was administered orally every day. It is now recognized that a deficit of mitochondrial energy metabolism  plays a role in the pathogenesis of chronic migraine headaches . Niacin Niacin might also prevent tension-type headaches by improving mitochondrial energy metabolism within the skeletal muscles, and by increasing blood flow and oxygenation to the skeletal muscles. The overall net-effect could be a reduction in lactic acid concentrations, leading to reduced episodes of muscular tension and soreness. Niacin may reduce lactic acid concentrations since supplemental niacinamide (the amide of niacin) has been shown to reduce blood lactate and pyruvate concentrations by more than 50% in a patient with MELAS  syndrome by the third day of treatment . This poThe side effects of intravenous niacin were found to be minimal from the summarized articles. The most common side effects were abdominal cramping, vomiting, and uncomfortable sensations of the skin and burning. A few of the patients described found the treatments to be worse than their headaches. In the articles where blood pressures were evaluated, little-to-no change occurred among the individuals treated with intravenous niacin. In a more recent study, parenteral niacin given to hypertensive and normotensive patients demonstrated a significant decrease in systolic, diastolic, and pulse pressures among the hypertensive subjects .With respect to the oral administration of niacin, very few patients reported side effects. Even though niacin was well tolerated orally, we previously reported in a randomized placebo-controlled trial examining the safety of immediate-release or crystalline niacin, that it can be associated with intolerable side effects . The mosThe side effects of greater concern from oral niacin have to do with sustained- or slow-release preparations. These preparations are better in terms of compliance since patients experience less cutaneous flushing with them. However, the use of these preparations have been associated with reversible hepatic toxicity in doses equal to or greater than 1500 mg per day with an acute onset of clinical symptoms of hepatitis in a relatively short period of time (2 days to 7 weeks) . Other rEven though niacin's mechanisms of action have not been substantiated from controlled clinical trials. It is possible that this agent has a beneficial effect upon migraine and tension-type headaches and the prophylaxis of these headaches. It is imperative that properly designed controlled trials are developed in order to determine niacin's true therapeutic effects and adverse effect profile. In terms of its ability to abort acute migraine headaches, a placebo-controlled trial of parenteral niacin and sumatriptan seems to be worthy of consideration. In terms of prophylaxis, a placebo-controlled trial of oral niacin and riboflavin or coenzyme Q10 also seems worthy of investigation.Dr. Jonathan Prousky is associated with Swiss Herbal Remedies, Ltd., a company that sells nutritional supplements including niacin. They had knowledge of this manuscript and have not seen this manuscript.JP wrote the manuscript. DS contributed to the text, revised the results section, and assisted with the tables.


This study was carried out to assess the reliability of core biopsy in predicting the grade and type of cancer accurately as obtaining this information can influence initial therapeutic decisions.Core biopsy is a method of choice for the triple assessment of breast disease as it can reliably distinguish between benign and malignant tumours, between kappa statastics.A total of 105 patients who had invasive breast carcinoma diagnosed by core biopsy in year 2001 and who subsequently underwent surgical management were included. The core biopsy results were compared with final histology with the help of A moderate level of agreement between the predicted grades and final grades was noted (kappa = 0.585). The agreement was good between predicted and final type of tumour (kappa = 0.639).Core biopsy as a predictor of grade and type has limited use at present. We suggest that initial clinical decisions should not be based on the results of core biopsy. Collins et al have shown that majority (83%) of core biopsies and excisional procedures demonstrate exact histological agreement [Core biopsy is rapidly replacing fine needle aspiration cytology (FNAC) as a procedure of choice for the triple assessment of the breast problems. Where there is access to an experienced cytopathologist, the FNAC can provide a rapid and cost effective means of triage of patients who would benefit from more expensive core biopsy . Core bigreement . Core bigreement .As the range of options for the treatment of the breast cancer widens, it has become increasingly important that clinicians are provided with accurate prognostic information to base the initial therapeutic decisions on. Prognostic factors for breast cancer have been extensively studied. Histological grade and type can be used to predict biological behaviour as has been assessed by overall survival and local recurrence for women with primary breast carcinoma -9. HistoThe aim of this study, therefore, was to see how reliable core biopsy is in predicting the grade and the type of cancers, as that could influence the further management of breast cancer.in-situ diagnosed by core biopsy and patients who underwent neo-adjuvant therapy were excluded from the study. Level of agreement between core and excision biopsy was assessed using kappa statistics.All patients with invasive breast cancer diagnosed by the core biopsy and treated subsequently by surgical excision in the year 2001, at a district general hospital were included in the study. Of the 105 patients whose records were studied retrospectively, 47 lesions were palpable and 58 lesions were screen detected. The core biopsies were performed under ultrasound guidance as a part of triple assessment and at least four cores were obtained from palpable lesions and six or more from screen detected lesions with a 22 mm automated core biopsy gun. Two dedicated breast pathologists had authorised all the reports. Age of patients ranged from 35 to 84 with a median age of 62 years. The histology reports for the core biopsy and final histology were extracted and compared. Carcinoma Of 105 patients there was no prediction of grade in 2 patients and in 19, a prediction of grade 1 or 2 or grade 2 or 3 was made. This left 84 where a clear prediction was made. On final histology 35 (33.3%) were categorised as grade I, 45 (42.8%) were grade II and 25 (23.8%) were grade III. The predicted grades versus final grade results are detailed in table Of the 84 cores in which clear prediction of grade was made 63 (75%) were correct. All 21 of grade 1's were predicted correctly, 35 (71%) of grade 2's were predicted correctly but only 7 (50%) or grade 3's were predicted correctly on core biopsy. Of the predicted grade 2's which were reclassified, 5 (10%) were downgraded and 9 (18%) were upgraded. Of the reclassified grade 3's, 6 (43%) were downgraded to grade 2 and 1 (7%) was downgraded to grade 1.Of 105 patients, 101 patients had a prediction of type made. Of 84 cases predicted to be ductal, 81 (96%) were correct and one case was reclassified as mixed histology. Of the 14 predicted to be lobular 9 (64%) were correct and one reclassified as mixed Table . Of the In general the level of agreement between the predicted grades and final grades was moderate (kappa = 0.585) and between predicted and final types was slightly better (kappa = 0.639).et al reported percutaneous, image guided biopsy to be an accurate diagnostic alternative to surgical biopsy in women with mammographically detected suspicious breast lesions [Fajardo  lesions . The fal lesions . However lesions  or amoun lesions  has not  lesions .Histological grade and type, tumour size and presence or absence of axillary node metastases is well-recognised prognostic factors of breast cancer. Tumour grade, size and nodal involvement are three factors considered in Nottingham Prognostic Index . HistoloGreen Hough 1925) was the first to categorise the breast tumours into three grades according to its differentiation. He also assessed the association of grades with "cure" though the term cure was not clearly defined . Since t was the Of the two major histological types, lobular is known for its multifocality and multicentricity and its diffusely infiltrating nature . It is iOur results suggest that the prediction of grade and type of breast cancer from core biopsy has only limited use at present. The group of patients we would like to be predicted most accurately would have been those with a high-grade and lobular type, for the reasons stated above. Our results suggest that these patients are most difficult to predict in practice. However, present study being retrospective has its own drawbacks. A prospective study specifically aimed at Kappa statistics between core biopsy and final histopathology may be able to answer this question better. Further refinements are needed in technique of core biopsy and these technical innovations will ultimately improve the results of core biopsy.The author(s) declare that they have no competing interests.AD: Original idea, planning of study, background search, data compilation and drafting the manuscript.TG: Data collection, data compilation, help with the manuscript drafting.SH: Overall supervision and guidance with the study, helped in the analysis and helped with manuscript drafting and revisions.All authors read and approved the final versionNone


Rare mutations of the epithelial sodium channel (ENaC) result in the monogenic hypertension form of Liddle's syndrome. We decided to screen for common variants in the ENaC βand γ subunits in patients with essential hypertension and to relate their occurrence to the activity of circulating renin-angiotensin-aldosterone system.Initially, DNA samples from 27 patients with low renin/low aldosterone hypertension were examined. The DNA variants were subsequently screened for in 347 patients with treatment-resistant hypertension, 175 male subjects with documented long-lasting normotension and 301 healthy  Plasma renin and aldosterone levels were measured under baseline conditions and during postural and captopril challenge tests.Xenopus oocytes, neither of the two ENaC amino acid-changing variants showed a significant difference in activity compared with ENaC wild-type. No direct evidence for a mRNA splicing defect could be obtained for the βENaC intronic variant. The ratio of daily urinary potassium excretion to upright and mean  plasma renin activity was higher in variant allele carriers than in non-carriers (p = 0.034 and p = 0.048).Two commonly occurring βENaC variants (G589S and a novel intronic i12-17CT substitution) and one novel γENaC variant (V546I) were detected. One of these variants occurred in a heterozygous form in 32 patients, a prevalence (9.2%) significantly higher than that in normotensive males (2.9%, p = 0.007) and blood donors (3.0%, p = 0.001). βENaC i12-17CT was significantly more prevalent in the hypertension group than in the two control groups combined (4.6% vs. 1.1%, p = 0.001). When expressed in At least 9% of Finnish patients with hypertension admitted to a specialized center carry genetic variants of β and γENaC, a three times higher prevalence than in the normotensive individuals or in random healthy controls. Patients with the variant alleles showed an increased urinary potassium excretion rate in relation to their renin levels. Epidemiological studies have shown a significant correlation of blood pressure levels in close relatives and higher concordance values for occurrence of hypertension in monozygotic vs. dizygotic twins, and thus support the idea that genetic factors influence susceptibility to essential hypertension . While rDisappointments in the previous strategies justify alternative approaches in which a better phenotyping of the study individuals is connected to their targeted molecular genetic characterization. There are several features that collectively make the genes encoding the beta (βENaC) and gamma (γENaC) subunits of the kidney tubular epithelial sodium channel as serious candidates for susceptibility genes of low-renin human essential hypertension. First, gain of function mutations in β and γ ENaC subunits cause Liddle's syndrome, a well-known monogenic form of human hypertension associated with low renin activity and low plasma aldosterone level -15. SecoThe clinical records of all consecutive patients with hypertension n = 615) referred to the Hypertension Outpatient Ward, Helsinki University Central Hospital, between 1992–96 were reviewed. Moderate-to-severe hypertension, suspicion of secondary forms of hypertension, or hypertension resistant to drug treatment were causes to the admittance. A letter with request to donate a blood sample for genetic studies on hypertension was sent to those 598 individuals whose address became available in 1998. A total of 399 individuals (67%) of these responded and were subsequently examined at the Hypertension Outpatient Ward in 1998 to 1999. Clinical and family histories were recorded, and venous blood samples taken for DNA analysis. Based on the previous documents and current examinations, altogether 52 individuals were excluded from the present study: clinical records were missing or insufficient in four cases, 22 subjects were considered as normotensive, while 26 were judged to have a secondary form of hypertension. The latter group consisted of the following cases: renal artery stenosis (n = 12), adrenal cortical adenoma (n = 3), hydronephrosis (n = 2), pheochromocytoma (n = 1), IgA glomerulonephritis (n = 1), non-specific chronic glomerulonephritis (n = 1), LED nephritis (n = 1), diabetic nephropathia (n = 1), chronic pyelonephritis (n = 1), hypernephroma (n = 1), fibromuscular dysplasia (n = 1), unspecified renal failure (n = 1). The remaining 347 patients  comprised our final cohort of patients with moderate-to-severe essential hypertension. Antihypertensive drug treatment was in use in 283 (82%) of the patients . At least two concomitant drugs were used by 24% of the patients. A flow-chart of the study design is illustrated in Fig. 5 referreDNA was extracted from 301 randomly selected healthy blood donors aged 40–50 years (mean, 45 years) visiting the Finnish Red Cross Blood Transfusion Service. Their residences represent the same capital area from which the hypertensive patients originated.These individuals were selected from the participants in the Alpha-Tocopherol, Beta Carotene (ATBC) study  using thThe Ethics Review Committee of the Helsinki University Central Hospital approved this study, and all patients and controls gave their informed consent.The patients were advised to stop using estrogens and spironolactone at least 4 weeks before the tests, diuretics and prostaglandin inhibitors at least two weeks before the tests, and β-adrenergic antagonists and ACE inhibitors at least one week before the test. The only antihypertensive agents permitted at the time of the test were calcium channel blockers. Some of the patients were on oral potassium supplementation because of hypokalemia. The mean baseline blood pressure level at the time of captopril test was 139 ± 16/94 ± 10 mmHg in those without any drugs (n = 79), and 142 ± 16/95 ± 11 mmHg in those with calcium channel blockers (n = 234).Fasting blood samples were taken for determination of serum creatinine, uric acid, cholesterol, potassium, sodium and blood glucose concentrations. Urine samples for determination of the daily (24 h) excretion of potassium and sodium were collected. Most hypertensive patients (n = 298) underwent a test for the responsiveness of serum aldosterone level and plasma renin activity to postural change. To this end, the first blood sample was taken after at least 60 minutes of rest in supine position. After 2 hours of standing and moderate walking, a second blood sample was taken. This test was carried out at the inpatient ward in 220 cases and at the outpatient ward in 78 cases. Urinary electrolyte excretion rates were analyzed in 262 patients (26 ENaC variant carriers and 236 non-carriers) who did not use potassium supplementation.One to three days later, a captopril challenge test (CCT) was carried out as described earlier . This teGenomic DNA was extracted from peripheral venous blood using standard techniques. For targeted search for ENaC variants postulated to be associated with increased channel activity, we chose to sequence the exons 13 coding for the carboxyterminal domains of βENaC (amino acids 515–640) and γENaC (amino acids 524–649), as well as the 5'-flanking intronic regions, using oligonucleotide primers, PCR (polymerase chain reaction) conditions and sequencing instruments described previously by us . DNA samAluI (New England Biolabs, Beverly, Massachusetts, USA), followed by analysis of the cleavage products on a 12% polyacrylamide gel. The wild-type (wt) allele results in longest fragments of 266 and 137 bp, while the variant allele produces fragments of 266 and 147 bp for βENaC -i12 -17CT, and 240 and 137 bp for βENaC G589S. For the γENaC V546I variant, 2.0 U of SfaNI (New England Biolabs) was used and the cleavage products were analyzed on a 2% agarose gel. The resulting fragment sizes were 357 and 77 bp for the wild-type allele and 279, 78 and 77 bp for the variant allele.Specific PCR-based methods were set up for assaying the three ENaC variants detected during the present study. After PCR of the βENaC fragment, the βENaC -i12 -17CT and βENaC G589S variants could be assayed simultaneously. An aliquot (8 μl) of the PCR product was digested with 3.0 U of in silico using GrailEXP v3.3  exon prediction program [For studies on the possible splicing errors brought about by the βENaC i12-17 variant, we collected lymphocytes from two subjects heterozygous for this variant and one control subject. Total lymphocytic RNA was isolated using Qiagen RNeasy kit , and first strand synthesis was performed using Superscript system for RT-PCR . For gene-specific PCR, we used two sets of primers amplifying a fragment extending from exon 12, either to exon 13 (180 bp) or 141 bp downstream of exon 13 (551 bp). The amplified products were run on a 12 % polyacrylamide gel and visualized by ethidium bromide. The amplified fragments were also sequenced to exclude presence of any splicing defects. Additionally, possible splicing differences between the wild-type and i12-17 variant of βENaC were studied  program .Agcttccagcctg-3' and 5'-gctgctctgttgtctgcAtcatcgagatcatcgagg-3' for the G589S and V546I mutations, respectively. The primer 5'-ccctcgctcgTgtgatctggt-3', which mutates the XhoI restriction enzyme site in the pBSK-SP6-globin vector, was used as the selection primer in the mutagenesis reactions. The mutagenic clones were sequenced to confirm the presence of the mutations and to exclude undesired errors during mutagenesis.The human βENaC cDNA and γENaC cDNA cloned into the pBSK-SP6-globin vector were used in construction of the βENaC G589S and γENaC V546I mutations, respectively. Site-directed mutagenesis was performed using the Transformer site-directed mutagenesis kit . Mutagenic primers used were 5'-cacaccaactttXenopus oocytes were injected with mRNAs encoding the β human (h)ENaC wild-type or βG589S hENaC mutant, the γhENaC wt or γV546I hENaC mutant together with the mRNA encoding the αhENaC wt. The total amount of mRNA encoding the three αβγ ENaC subunits was 10 ng. Electrophysiological measurements were taken at 16–24 hours after injection. ENaC activity was assessed by measurement of the amiloride-sensitive current (INa in μA) recorded at -100 mV with a two-electrode voltage clamp amplifier (TEV-200, Dagan Corp.) in a standard solution containing 110 mmol/L NaCl, 1.8 mmol/L CaCl2, 10 mmol/L HEPES-NaOH, pH 7.35. The amiloride concentration used was 5 μmol/l in the bath solution. Four batches of oocytes were obtained from different Xenopus frogs in which 5 to 7 oocytes were tested for each αβγ ENaC wt and ENaC variants.Healthy stage V and VI The renin and aldosterone values were nonnormally distributed, as analyzed using skewness, kurtosis and Kolmogorov-Smirnov tests. Therefore, nonparametric tests (Mann-Whitney's U) were used in the statistical analyses, and interquartile (IQ) range and median are used to describe the distributions of target variables. When covariates were included in the analyses, ANCOVA with ranks or logarithm-transformed values of the variables was used. Chi square test, or Fisher's exact test if observed frequency in any cell was less than five, were used for the frequency analysis of the variants. Logistic regression was used to obtain age and gender adjusted odds ratios for hypertension in ENaC variant carriers vs. non-carriers. All data was analyzed using statistical SPSS program (version 11.0). Because of relatively small variant group sizes, the primary analyses were performed with all variant groups combined. Secondarily, the variant groups were also compared separately with the wild-type ENaC group.DNA samples of 27 hypertensive patients with lowest renin activities and aldosterone concentrations were initially selected for targeted search for ENaC variants. The sequencing strategy chosen permits detection of mutations and polymorphisms in the entire coding parts of exons 13 of β and γENaC genes, as well as 26 or 43 nucleotides at the 3'-ends of introns 12. Three different single-nucleotide substitutions were detected, two in the βENaC and one in the γENaC subunit . Subanalysis of the three groups indicated that the variant allele frequency was higher among the hypertensive patients (9.2%) than in normotensive males  or blood donors , while in the latter two groups it was similar , normotensive males n = 175) and randomly chosen blood donors (n = 301)  in the two tests according to the ENaC variant carrier status. We found some evidence of a blunted renin response to both postural (p = 0.21) and captopril (p = 0.087) challenge tests in carriers of variant alleles compared to non-carriers, but there was wide interindividual variation in the test results Fig. . Use of We next related the activity of circulating renin-aldosterone system to sodium-potassium homeostasis in ENaC variant carriers and non-carriers. Serum sodium and potassium concentrations in these two groups of hypertensive patients were similar Table . UrinaryXenopus oocytes, the most commonly used expression system for ENaC functional studies. When αβγ ENaC subunits were co-expressed to obtain maximal channel activity, neither the βG589S nor γV546I affected ENaC activity as measured by the amiloride-sensitive Na+ currents. In other words, these data indicate that the current carried by Na+ ions through ENaC channels present at the cell surface is similar for ENaC wild-type and mutant channels, indicating that the βG589S and γV546I mutations have no detectable functional consequences on ENaC activity, at least when expressed in Xenopus oocytes.We tested whether the βG589S or γV546I have any functional impact on ENaC expressed in in silico analysis of the βENaC wild-type and variant DNA sequences suggested no differences in exon splicing. However, this analysis is only of predictive value and does not exclude a splicing defect introduced by the variant nucleotide in renal tissue.In order to clarify whether the C-T substitution at position -17 of the intron 12 of the βENaC could affect mRNA splicing, cDNA was synthesized from an RNA fraction prepared from lymphocytes of two hypertensive patients heterozygous for the βENaC i12-17CT mutation and a control individual without this gene variant. Primer pairs for reverse transcription were designed in a way permitting identification of a possible failure to splice intron 12 properly. Regardless of the primer pairs used, similar DNA fragments were generated from the samples of the βENaC i12-17CT carriers and control subject (data not shown). Furthermore, sequence analysis of the amplified DNA fragments revealed the presence of only normally spliced DNA sequence in the βENaC i12-17CT carriers. Furthermore, in vitro studies have failed to demonstrate a gain-of-function for these ENaC variants, their association with an increased urinary potassium loss in relation to existing plasma renin activity suggests that in the long run in vivo they may result in sodium retention, suppression of renin and aldosterone levels and hypertension.The present study indicates that three common variants of the kidney epithelial sodium channel ENaC occur approximately three times more often in patients with moderate-to-severe essential hypertension compared to normotensive males and  While direct in vitro, nor showed cosegregation with hypertension [A large number of common and rare polymorphisms of the α-, β-and γENaC have been described in different populations (reviewed in  and 27]27]), butrtension ,28. The rtension .Xenopus oocytes, these investigators were able to show a borderline 1.3 to 1.5-fold increase in activity for the G589S variant compared with the wild-type subunit. A similar trend was noticed in our experiments  and 175 normotensive males (p = 0.043). Patients with this variant allele displayed the lowest plasma renin levels and responses of all the subgroups examined .The authors declare that they have no competing interests.TH-H collected the clinical material, and participated in the DNA analyses and drafting of the manuscript. KK and TPH designed the study and drafted the manuscript. IT, TT, FF, KH designed the clinical chemical and hormonal assays, and participated in collection of the patient material. HF, HEM and KP participated in DNA analyses and bioinformatics. JV and TK collected the control populations and designed their studies. SS consulted in the statistical analyses and assisted in data handling. IG and LS carried out the electrophysiological studies and participated in drafting of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Human monoclonal antibodies (MAbs) are needed for colon cancer radioimmunotherapy (RIT) to allow for repeated injections. Carcinoembryonic antigen (CEA) being the reference antigen for immunotargeting of these tumors, we developed human anti-CEA MAbs.®-G2 animals were immunized with CEA. Among all the antibodies produced, two of them, VG-IgG2κ and VG-IgM, were selected for characterization in vitro in comparison with the human-mouse chimeric anti-CEA MAb X4 using flow cytometry, surface plasmon resonance, and binding to radiolabeled soluble CEA and in vivo in human colon carcinoma LS174T bearing nude mice.XenoMouse8 M-1 and 1.30 ± 0.06 × 108 M-1, respectively, as compared with 0.61 ± 0.05 × 108 M-1 for the reference MAb X4. In a soluble phase assay, the binding capacities of VG-IgG2κ and VG-IgM to soluble CEA were clearly lower than that of the control chimeric MAb X4. A human MAb concentration of about 10-7 M was needed to precipitate approximatively 1 ng 125I-rhCEA as compared with 10-9 M for MAb X4, suggesting a preferential binding of the human MAbs to solid phase CEA. In vivo, 24 h post-injection, 125I-VG-IgG2κ demonstrated a high tumor uptake (25.4 ± 7.3%ID/g), close to that of 131I-X4 (21.7 ± 7.2%ID/g). At 72 h post-injection, 125I-VG-IgG2κ was still concentrated in the tumor (28.4 ± 11.0%ID/g) whereas the tumor concentration of 131I-X4 was significantly reduced (12.5 ± 4.8%ID/g). At no time after injection was there any accumulation of the radiolabeled MAbs in normal tissues. A pertinent analysis of VG-IgM biodistribution was not possible in this mouse model in which IgM displays a very short half-life due to poly-Ig receptor expression in the liver.Flow cytometry analysis demonstrated binding of MAbs on CEA-expressing cells without any binding on NCA-expressing human granulocytes. In a competitive binding assay using five reference MAbs, directed against the five Gold CEA epitopes, VG-IgG2κ and VG-IgM were shown to be directed against the Gold 4 epitope. The affinities of purified VG-IgG2κ and VG-IgM were determined to be 0.19 ± 0.06 × 102 fragments, or as a bispecific antibody.Our human anti-CEA IgG2κ is a promising candidate for radioimmunotherapy in intact form, as F(ab') On thAlthough targeting of solid tumors with radiolabeled antibodies was first reported years ago ,5, RIT sColorectal cancers represent a high percentage of solid tumors and are dramatically in need of therapeutic progress. Surgery is the only potentially curative treatment. Despite recent developments in chemotherapy protocols, the overall median survival in metastatic colorectal cancer remains inferior to two years, and the recurrence rate after resection of a stage III tumor is up to 50% -18. For ® technology, we describe the generation and the characterization of two fully human anti-CEA antibodies, one IgG2κ and one IgM, designed for RIT of colorectal cancers.Different chimeric or humanized anti-CEA MAbs have been described and evaluated in experimental and clinical studies ,15,19,20®-G2 strain, engineered to produce fully Human IgG2κ antibodies, was described by Mendez et al. [®-G2 animals were immunized i.p. with 20 μg of human recombinant CEA (rhCEA) [Generation and characterization of the XenoMousez et al. . XenoMou (rhCEA)  emulsifi4κ subclass [1 [The mouse-human chimeric MAb X4 was used as positive control in all the experiments. MAb X4 was constructed using the variable domains from the murine MAb CE25 and the constant domains from a human IgGsubclass ,24. It isubclass . MAb F6 class [1 .The CEA-positive human colon carcinoma LS174T cell line  was obta®-1077 and HISTOPAQUE®-1119. Following centrifugation at 700 g for 30 minutes, cells of the granulocytic series were found at the 1077/1119 interphase.Granulocytes were obtained from heparin-treated human peripheral blood by using gradient density centrifugation methods. A double gradient was formed by layering an equal volume of HISTOPAQUEκ (Sigma, Lyon, France) and HRP-conjugated sheep anti-human IgG (γ chain)  were used as detection antibodies.The specificity of the antibodies in hybridoma supernatants was determined by ELISA using rhCEA to capture the antibodies (coating overnight at 2 μg/ml rhCEA at room temperature). Horse radish peroxidase (HRP)-conjugated goat anti-human IgG5 cells were incubated for 1.5 h at 4°C with hybridoma supernatants or controls (RPMI medium for background measurement and RPMI containing 20 μg/ml MAb X4 for positive control). After washing, the cells were incubated with an FITC conjugated goat anti-human IgG kappa light chain (Sigma) or with the murine anti-human μ chain DA4-4 (ATCC HB-57), FITC labelled in our laboratory, for 1 h at 4°C; then they were washed twice before analysis on a FACScanII (Becton-Dickinson, Le-Pont-De-Claix, France). Each figure represents data obtained from analysis of 10000 cells.Determination of CEA or NCA specific antibodies in hybridoma supernatants was carried out by flow cytometry using CEA positive cells (CO115-5F12) and NCA positive cells (human granulocytes). About 5 × 10® NHS-anti-human κ chain MAb HP6053 (ATCC CRLC-1758).The percentage of fetal calf serum was gradually reduced in the culture medium before MAb purification (10, 5, 2.5 and 0%). MAbs were purified from large volumes of hybridoma supernatants or ascites produced in nude mice using HitrapProteins separated on SDS-PAGE 6% polyacrylmamide gels were transferred to a nitrocellulose membrane (Protran BA85, Schleicher and Schuell, Dassel, Germany). Non-specific binding sites were blocked overnight at 4°C by incubation with 5% (w/v) non-fat dry milk in TBS. The membrane was probed for 2 h at room temperature with serum diluted 1:1000 or the following antibodies: anti-human κ light chain-HRP conjugate (A7164, Sigma), anti-human λ light chain-HRP conjugate (A5175, Sigma), anti-human μ chain-HRP conjugate (A0420, Sigma), rabbit anti-human J chain-specific antiserum . Bound sin silico methods.Antibody VH and Vκ cDNAs were recovered from hybridomas by RT-PCR and sequenced using the ABI-PRISM Big Dye Terminator Cycle Sequencing Kit (Perkin Elmer, Boston, MA). Determination of V, D, and J gene usage was performed using The affinities of the antibodies for CEA were determined by using surface plasmon resonance (SPR) technology . rhCEA  was immo125I or 131I, kindly provided by Schering-CIS Biointernational by the iodogen method (1,3,4,6-tetrachloro-3α, 6α-diphenylglycoluryl, Sigma). Free radioiodine was separated from the protein on a Sephadex G-25 column (Pharmacia) equilibrated in PBS, pH 7.4.Batches of 50 μg or 100 μg of MAb or rhCEA were labelled with 4.6 MBq or 11.5 MBq, respectively, of 125I-rhCEA  was incubated with increasing concentrations of antibody (68.3 × 10-12 to 33.3 × 10-15 M) for 2 h in 0.15 M phosphate buffer, pH 7.4. CEA-antibody complexes were precipitated at 4°C with 53.5% (v/v) saturated ammonium sulfate. Background binding to an irrelevant human IgG, MonoD, was subtracted. The radioactivity precipitated with a rabbit polyclonal anti-CEA serum was taken as 100%.About 1 ng 125I-human MAb (15 ng) was added to the wells and incubated for 1 h 30 at 37°C. The percentage of binding was determined by measuring the radioactivity bound to the rhCEA after two washings.A competitive binding assay of radiolabeled human MAb and unlabeled anti-CEA MAb was used to determine the CEA epitope recognized by the human MAbs. RhCEA (100 ng/well) in TBS was coated on microtiter wells overnight at room temperature. An excess (500 ng/well) of each of the different reference anti-CEA MAbs, specific for the Gold epitopes 1 to 5, was then added to the wells and incubated for 1.5 h at 37°C. Then, without washing, each 3 (100 to 300 mm3), mice were grouped according to tumor volume. Lugol iodine solution (10% solution) was added to the drinking water one day before the injection of radiolabeled MAbs. Groups of four mice were injected with a 125I-labeled human MAb (VG-IgG2κ or VG-IgM) together with 131I-labeled chimeric MAb X4 as positive control. The total amount of each injected antibody was adjusted to 8 μg protein by adding unlabeled MAb. To determine the biodistribution of the MAbs, mice were sacrificed 24 or 72 h after injection. The blood, tumor, and all normal organs were weighed, and the differential radioactivity was measured in a dual channel scintillation counter. The results are expressed as the percentage of the injected dose of radioactivity present per gram of tissue (% ID/g).Two million LS174T cells were grafted s.c. into the right flank of female Swiss nude mice (nu/nu, Iffa Credo, l'Arbresle, France). When the tumors had reached a volume of about 150 mm®-G2 animals were immunized with rhCEA. The XenoMouse®-G2 strain produces both fully human IgG2κ and fully human IgMκ antibodies as part of the normal immune response, as described by Mendez et al. [in vivo tumor targeting purposes. For this reason, (i) hybridoma supernatants were screened by flow cytometry because, by this technique, the selection is made on cell membrane-bound CEA, which is in a conformation as close as possible to that observed in vivo; and (ii) hybridoma supernatants were screened on human granulocytes to eliminate all hybridomas producing anti-NCA antibodies.In order to develop human anti-CEA MAbs, XenoMousez et al. . Fusion H and VL domains sequencing confirmed the VG-IgG2κ and VG-IgM monoclonality and the isotype (data not shown). Flow cytometry analysis demonstrated strong binding of purified MAbs on CEA-expressing cells CO115-5F12  as compared with 10-9 M for MAb X4 , very close to that of 131I-X4 (21.7 ± 7.2% ID/g) . Due to the mouse poly-Ig receptor expression in liver, 125I-VG-IgM was eliminated very rapidly, giving only 3.7 ± 1.0 and 0.3 ± 0.1% ID/g blood at 24 h and 72 h, respectively. This short clearance was responsible for a low tumor uptake (7.4 ± 2.8 and 1.8 ± 2.4% ID/g tumor at 24 h and 72 h, respectively). The percentage of injected MAb recovered in the whole mouse at 24 h was only 20.5% for 125I-VG-IgM as compared with 53.7% for 131I-X4.In two other groups of mice, Monoclonal antibodies are now routinely used in the clinic. Whereas the first generation of MAbs were murine or chimeric antibodies, it is now clear that the best clinical results are obtained with humanized or fully human MAbs . Fully hIn some situations, naked MAbs have demonstrated their therapeutic efficacy, particularly, when the target antigen is a receptor implicated in cell proliferation processes ,34. Howein vitro and presented preliminary data on tumor growth inhibition using MKN-45 cells grafted into SCID mice. However, they did not give any results on the biodistribution of their antibodies in radiolabeled form nor indications on how they could be used in RIT. Anti-CEA MAb PR1A3, which exhibits preferential binding to cell-bound CEA, was recently humanized, but to our knowledge, this MAb has not yet been evaluated in experimental or clinical studies [131Iodine or 90Ytrium, in heavily pre-treated patients with metastatic colorectal cancer (MCRC) CT84.66 [Among solid tumors, colorectal cancers represent one of the main causes of death, and CEA is well known as an ideal target antigen for RIT of these cancers . Up to n studies . A few c CT84.66 ,15. One In the present study, the characterization of our fully human anti-CEA MAbs was conducted in comparison with the chimerized anti-CEA MAb X4, which has been shown to be clinically relevant ,24. Furtin vivo use in patients where some circulating CEA can be found. The molecular basis of this observation is not clear. The only possible comparison is with MAb PR1A3, which preferentially binds to cell bound CEA, and to a recombinant chimeric protein containing only the CEA B3 domain [The VG-IgG2κ and VG-IgM affinity constants were found to be similar to that of the control MAb X4 when determined using the BIACORE technology, but the binding of the human MAbs to soluble CEA Figure  was clea3 domain ,38. This3 domain .125I-VG-IgG2κ localized in the tumor up to 26.2 ± 1.7% ID/g as compared with 28.5 ± 2.8% ID/g for 131I-35A7, suggesting a tumor residence time as long as that observed for MAb 35A7 (data not shown). These results could seem contradictory, given the fact that the affinity of VG-IgG2κ for CEA is not as high as that of Mab 35A7. This could be explained by the "affinity barrier" effect described in solid tumors by several authors who demonstrated that very high affinity MAbs localized at the periphery of tumor nodules and that lower affinity MAbs are able to distribute homogenously in these nodules [Using human colon carcinoma bearing nude mice, we obtained high tumor uptakes with VG-IgG2κ, making this antibody a good candidate for future clinical studies Figure . In addi nodules -43.Since, up to now, there are no data available on the biodistribution of any anti-CEA IgM in mice, we decided to analyze the biodistribution of our VG-IgM in LS174T tumor bearing nude mice. The disappointing tumor uptakes (7.4 ± 2.8 and 1.8 ± 2.4%ID/g tumor at 24 h and 72 h, respectively) could be attributed to a very short half-life due to poly-Ig receptor expression in the mouse liver which induces a rapid hepatobiliary transport of poly-IgA and IgM ,45. Base2 fragments, or as a bispecific antibody to be used in the affinity enhancement system (AES) approach [In the present study, we described two fully human anti-CEA MAbs. Even though the results obtained in tumor bearing nude mice cannot be extrapolated directly to humans, VG-IgM remains attractive for RIT of CEA-positive peritoneal carcinomatosis in man since humapproach . Furtherapproach , and it approach ,52 and fapproach .No competing interest for VG, FG, EF, MY, MP and AP. RXL and BB are employees of Abgenix, Inc.in vivo experiments. FG and EF participated in the cell culture experiments and antibody characterization assays. MP performed the affinity measurements using BIACORE. MY participated in the design of the study. RXL and BB sequenced the VH and VL domains. AP conceived the study, participated in its design and coordination. All authors read and approved the final manuscript.VG participated in the design of the study, performed all the cell fusions, cell culture experiments, antibody characterization assays and The pre-publication history for this paper can be accessed here:


Syzygium aromaticum (L.) Merr. & Perry. (clove) has been used in Unani medicine since ancient times for the treatment of male sexual disorders. The present study is aimed to investigate the effect of 50% ethanolic extract of clove on general mating behaviour, libido, potency along with its likely gastric ulceration and adverse effects on sexually normal male albino rats.The flower bud of The suspension of the extract was administered orally at the dose of 100, 250, and 500 mg / kg, to different groups of male rats (n = 6) once a day for seven days. The female albino rats involved in mating were made receptive by hormonal treatment. The general mating behaviour, libido and potency were determined and compared with the standard reference drug sildenafil citrate. The probable gastric ulceration and adverse effects of the extract were also evaluated.Oral administration of the extract significantly increased the Mounting Frequency, Intromission Frequency; Intromission Latency, Erections; Quick Flips, Long Flips as well as aggregate of penile reflexes and caused significant reduction in the Mounting Latency and Post Ejaculatory Interval. The most appreciable effect of the extract was observed at the dose of 500 mg/kg. The test drug was also found to be devoid of any conspicuous gastric ulceration and adverse effects.The results indicated that the 50% ethanolic extract of clove produced a significant and sustained increase in the sexual activity of normal male rats, without any conspicuous gastric ulceration and adverse effects. Thus, the resultant aphrodisiac effectivity of the extract lends support to the claims for its traditional usage in sexual disorders. Syzygium aromaticum (L.) Merr. & Perry. (Family: Myrtaceae) an evergreen tree 10–20 m in height indigenous to India, Indonesia, Zanzibar, Mauritius and Sri Lanka [Herpes simplex [Clove is the dried flower bud of ri Lanka ,4, carmiri Lanka ,6. It isri Lanka  and usefri Lanka . Clove iri Lanka . It inhi simplex . Phytoch simplex , phenyl  simplex , β caryo simplex , eugenol simplex . Eugenol simplex , and has simplex  and smoo simplex . Phyto c simplex , showed The present study thus, is aimed to investigate the aphrodisiac effect of 50% ethanolic extract of clove in detail, using multiple parameters along with its probable gastric ulceration and adverse effects in sexually normal male albino rats. The doses used in the study were selected according to the Freirich , multiplS. aromaticum (clove) was procured from the market (Delhi, India). A voucher (S721) sample was kept for further reference. The clove was crushed to coarse powder and sieved through No. 20 mesh size. The extraction was carried out by mixing the powdered clove with 1:3 w/v in 50% ethanol v/v by Soxhlet apparatus for 6 h. The extract was filtered and the solvent from the filtrate was removed by rotary evaporator under reduced pressure and low temperature. The yield of extract was 10.40% w/w in terms of dried starting material. It was yellowish and of pleasant smell. The extract was preserved in a refrigerator.The authenticated dried flower bud of Sildenafil citrate was purchased from Zydus Cadila, (Ahmadabad, India). Other drugs used were ethinyl oestradiol , progesterone  and 5% xylocane ointment ad libitum throughout the study except during the experiment. The study design was approved by the ethical committee of the Department for animal care and use.Twelve weeks old male and female albino rats of wistar strain weighing 350–400 g and 225–275 g respectively, were used for the study. They were housed singly in separate standard cages and maintained under standard laboratory conditions (temperature 24–28°C, relative humidity 60–70%, 12 h light-dark cycle) with free access to solid pellet diet (Gold Mohar, Lipton-India) and water Since clove in Unani medicine is orally administered, therefore, the extract of clove was suspended in distilled water using Tween 80 (1%) for oral administration. Sildenafil citrate and ethinyl oestradiol were also suspended in distilled water using Tween 80 (1%) separately, for oral use. Progesterone was dissolved in olive oil for subcutaneous injection. All the drug solutions were prepared just before administration.The test was carried out by the methods of Dewsbury and Davis Jr  and Szecth day after commencement of the treatment of the male animals. The experiment was conducted at 20:00 h in the same laboratory and under the light of same intensity. The receptive female animals were introduced into the cages of male animals with 1 female to 1 male. The observation for mating behaviour was immediately commenced and continued for first 2 mating series. The test was terminated if the male failed to evince sexual interest. If the female did not show receptivity she was replaced by another artificially warmed female. The occurrence of events and phases of mating were called out to be recorded on audio-cassette as soon as they appeared. Their disappearance was also called out and recorded. Later, the frequencies and phases were determined from cassette transcriptions: number of mounts before ejaculation or Mounting Frequency (MF), number of intromission before ejaculation or Intromission Frequency (IF), time from the introduction of female into the cage of the male upto the first mount or Mounting Latency (ML), time from the introduction of the female up to the first intromission by the male or Intromission Latency (IL), time from the first intromission of a series upto the ejaculation or Ejaculatory Latency (EL), and time from the first ejaculation upto the next intromission by the male or Post Ejaculatory Interval (PEI). In the second mating series only the EL was recorded. The values for the observed parameters of the control, test and standard animals were statistically analysed by using one-way analysis of variance (ANOVA) method.The female animals were artificially brought into oestrus (heat)  by the Sth day at 20:00 h. The penis was exposed by retracting the sheath and 5% xylocaine ointment was applied 30, 15 and 5 min before starting observations. Each animal was placed individually in a cage and the receptive female rat was placed in the same cage. The number of mountings was noted. The animals were also observed for intromission and ejaculation. The MF in control, test and standard animals was statistically analysed by employing one-way analysis of variance (ANOVA) method.The test was carried out by the method of Davidson , modifieth day, the test for penile reflexes was carried out by placing the animal on its back in a glass cylinder partial restraint. The preputial sheath was pushed behind the glans by means of thumb and index finger and held in this manner for a period of 15 min. Such stimulation elicits a cluster of genital reflexes. The following components were recorded: Erections (E), Quick Flips (QF), and Long Flips (LF). The frequency of these parameters observed in control, test and standard groups was statistically analysed by using one-way analysis of variance (ANOVA) method.The effect of the test drug was studied according to the methods described by Hart and Haugen  and Hartth day all the animals were killed and the stomach was then incised along the greater curvature and washed carefully with physiological saline. Any gastric lesions were observed immediately using a magnifying glass. The number of erosions per stomach was assessed for severity, according to the score of Cioli et al [The male animals (350–400 g) were divided into 4 groups of 6 animals each. Group 1 represented the control group, which received 10 ml/kg of distilled water. Groups 2–3 received suspension of the extract orally at the doses of 100, 250 and 500 mg/kg, respectively, daily for 7 days. After the treatment, on 8li et al  : (o) abAll treated rats were observed at least once daily for any overt sign of toxicity , stress  and changes in behaviour (such as spontaneous movement in the cage, climbing, cleaning of face). In addition food and water intake were noted.The significance of difference between the means was determined by one-way analysis of variance (ANOVA) with post-hoc't' test. P value <0.05 was considered as significant.1) (P < 0.0001) and decreased Mounting Latency (ML) (P < 0.0001), Intromission Latency (IL) (P < 0.01) in a significant manner. The dose 250 mg / kg of the extract significantly increased the MF (P < 0.001), IF (P < 0.05) and significantly reduced the ML (P < 0.001), EL1 (P < 0.05); Post Ejaculatory Interval (PEI) (P < 0.001) and did not significantly alter the IL (P < 0.61), Ejaculatory Latency in second series (EL2) (P < 0.41). Whereas the dose of the test drug at 100 mg/kg significantly increased the MF (P < 0.05), PEI (P < 0.05) but did not significantly affect the IF (P < 0.61), ML (P < 0.61), IL (P < 0.61); EL1 (P < 0.61); EL2 (P < 0.81). However, the standard drug increased the MF (P < 0.0001), IF (P < 0.0001); EL1 (P < 0.0001), EL2 (P < 0.0001); PEI (P < 0.0001) as well as decreased ML ((P < 0.0001) and IL (P < 0.0001) in a highly significant manner (Table The data obtained with the mating behaviour test indicated that the clove-extract at the dose of 500 mg/kg increased the Mounting Frequency (MF) P < 0.0001), Intromission Frequency (IF) (P < 0.001) ; Ejaculatory latency in first series (EL, IntromiThe test for libido showed that the extract at the dose of 500 mg/kg increased the Mounting Frequency (MF) in a significant manner (P < 0.001). The extract at the doses of 100 mg/kg and 250 mg/kg did not significantly alter the MF(P < 0.24, P < 0.75 respectively). The standard drug strikingly increased the MF (P < 0.0001). Intromission and Ejaculation were found absent in control, test and standard groups (Table The test for potency exhibited that the higher dose (500 mg/kg) of the test drug significantly increased the frequency of Erections (E) (P < 0.0001), Quick Flips (QF) (P < 0.0001), Long Flips (LF) (P < 0.0001) as well as the aggregate of these penile reflexes (TPR) (P < 0.0001). The extract at the dose of 250 mg/kg significantly increased the E (P < 0.01), LF (P < 0.05) and TPR (P < 0.05) but comparatively less than the higher dose of the extract and standard drug, and did not significantly increase the QF (P < 0.10). whereas, the test drug at the dose of 100 mg/kg did not alter the E (P < 0.78), QF (P < 0.78); LF (P < 0.44) and TPR (P < 0.44) in a significant manner Table .Seven days treatment of low and high doses of the extract caused no significant ulceration in gastric mucosa of albino rats. Moreover, there were neither treatment related defects nor overt clinical signs of toxicity, stress or changes in behaviour and appearance evident. The food and water intake of all test drug treated rats remained similar to those of the control group.S. aromaticum) was tested in animal experimentation for its effect on sexual behaviour, and sildenafil citrate was used as the standard referent.In the present study, clove (1) and in second series (EL2) was studied. The test drug significantly increased the EL1 and EL2 as compared to control animals, whereas a highly significant increase was observed with the standard drug.The study showed that the 50% ethanolic extract of clove possesses significant sexual function enhancing activity as observed in sexual behaviour tests. Mating behaviour test revealed that the test drug significantly increased the Mounting Frequency (MF) and Intromission Frequency (IF) as compared to control but less than that of the standard drug. The (MF) and (IF) are considered as the indices of both libido and potency. So, this is an indication that the test drug possesses a sexual function improving effect. The premature ejaculation is one of the important causes of sexual dysfunction, so the assessment of Ejaculatory Latency in first series (ELThe test drug was found to produce a significant reduction in the Mounting Latency (ML) and Intromission Latency (IL) as compared to control while a highly significant decrease was found in ML of animals treated with sildenafil citrate. This is also an evidence of the sexual function improving effect of the clove.The Post Ejaculatory Interval (PEI) is considered as an index of potency and libido, and also a parameter of the rate of recovery from exhaustion after first series of mating. PEI was found significantly decreased with clove-extract and also with the standard drug. The test drug decreased PEI either by enhancing the potency and libido or by producing lesser exhaustion in the first series of mating or by both.The effect of the test drug on libido was also evaluated by testing the MF after genital anaesthetization which does away with the reinforcing effect of genital sensation thus affording the study of pure libido or intrinsic sexual desire. During the experiment the extract produced a significant increase in the MF of sexually normal male rats whereas the efficacy of the standard drug, as expected, was found to be highly significant. The MF was much reduced in control, test and standard animals in comparison with the MF of corresponding groups in mating behaviour test where the penis had not been anaesthetized. None of the control, test and standard animals were observed to show Intromission or Ejaculation because their occurrence depends upon local genital sensation which was obstructed due to anaesthetization.The effect on potency was also evaluated by testing the effect of the drug on the frequency of penile reflexes namely Erections (E), Quick Flips (QF), and Long Flips (LF). The test drug significantly increased the frequency of all the components of penile reflexes (E, QF, & LF) in the test animals as compared to control group but comparatively lesser than the standard drug. The aggregate of penile reflexes (TPR) was also found increased in the animals treated with the extract and sildenafil citrate. Therefore, the experiment revealed that the test drug produced a marked increase in potency.The spices are reported to produce an increase in gastric acid secretion by a cholinergic mechanism , and so,The results of the present study clearly proved that the clove is endowed with sexual function improving activity. This is in consonance with our earlier study showing sexual function improving effect of the test drug in male mice. However, the established drug i.e. sildenafil citrate exhibited, as expected, tremendous activity. With regard to the mechanism of the test drug, it is difficult to interprete the mechanism involved in potentiation of sexual function. The drugs induced changes in neurotransmitter levels or their action at cellular levels could change sexual behaviour . Hence, Moreover, research should be aimed at isolating the active principle(s) responsible for aphrodisiac activity and the mechanism by which the drug enhances sexual function. In addition, to discover the applied effective concentration or dosages of the extract, more studies are also required.The present results indicated that the 50% ethanolic extract of clove possesses potent aphrodisiac activity in normal male albino rats without any gastric ulceration and adverse effects and provided scientific evidence in favour of the claims made in Unani medicine that the clove is clinically useful as sexual invigorator in males.The author(s) declare that they have no competing interests.T-Supervised the design and coordinatrion of the study.SA-Practically conducted the design of the study.AL-Participated and performed the statistical analysisIA-Participated in the drafting of manuscript.All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


We show that a 2′-O-methyl oligonucleotide complementary to an siRNA can block mRNA cleavage in Drosophila embryo lysates and HeLa cell S100 extracts and in cultured human HeLa cells. In Caenorhabditis elegans, injection of the 2′-O-methyl oligonucleotide complementary to the miRNA let-7 can induce a let-7 loss-of-function phenocopy. Using an immobilized 2′-O-methyl oligonucleotide, we show that the C. elegans Argonaute proteins ALG-1 and ALG-2, which were previously implicated in let-7 function through genetic studies, are constituents of a let-7-containing protein–RNA complex. Thus, we demonstrate that 2′-O-methyl RNA oligonucleotides can provide an efficient and straightforward way to block small RNA function in vivo and furthermore can be used to identify small RNA-associated proteins that mediate RNA silencing pathways.Hundreds of microRNAs (miRNAs) and endogenous small interfering RNAs (siRNAs) have been identified from both plants and animals, yet little is known about their biochemical modes of action or biological functions. Here we report that 2′ O-methyl RNA oligonucleotides can block small RNA function in vivo - providing a tool to determine the biological functions of siRNA and microRNA. These oligonucleotides can also be used to identify small RNA-associated proteins that mediate silencing pathways2′- The endoribonuclease Dicer produces two types of small regulatory RNAs that regulate gene expression: small interfering RNAs (siRNAs) and microRNAs (miRNAs) . In animDrosophila S2 cells . RNAi is a powerful method for the study of gene function in animals and plants and has even been proposed as a therapy for treating genetic disorders and viral infections. Biochemical studies in S2 cells  and affiS2 cells  or immunS2 cells  from culfor RNAi a, 2002b.for RNAi .C. elegans, the miRNAs lin-4  luciferase (Pp-luc) mRNA target  strand of the siRNA duplex, thereby blocking incorporation of the guide (antisense) strand into RISC  to the target RNA than a 2′-O-methyl oligonucleotide. These data suggest that specific proteins in the RISC facilitate either target finding, target binding, or both. Consistent with this idea, inhibition of RISC function was incomplete using 21-nt 2′-O-methyl oligonucleotides (data not shown). Thus, target sequence flanking the site of complementarity to the siRNA guide strand may play a role in target RISC binding. Perhaps an active mechanism that involves target sequences flanking the siRNA facilitates the search for the target sequence; future studies will clearly be needed to test this idea.Thus, the interaction of 2′--O-methyl oligonucleotides are stoichiometric, irreversible, sequence-specific inhibitors of siRNA function in RNAi reactions using Drosophila embryo lysate. Can 2′-O-methyl oligonucleotides block siRNA function in vivo? To address this question, we carried out sequential transfection experiments using 1 nM, 5 nM, 10 nM, or 25 nM siRNA duplex. siRNA was transfected on the first day; then, reporter and control plasmids were cotransfected together with various amounts of 2′-O-methyl oligonucleotide on the second day. Silencing of Pp-luc, relative to the sea pansy (Renilla reniformis) luciferase (Rr-luc) control, was measured on the third day. For each siRNA concentration, we determined the concentration of 2′-O-methyl required for half-maximal inhibition of RNAi . The data fit well to a sigmoidal curve, consistent with the idea that, at these concentrations, increasing amounts of siRNA do not produce a corresponding increase in RISC activity. Higher concentrations of siRNA were not examined because they produce sequence-independent changes in gene expression  As a control, we also tested whether the oligonucleotide could block the activity of a let-7-containing RISC assembled in vitro in Drosophila embryo lysate. Addition of this 2′-O-methyl oligonucleotide efficiently blocked target RNA cleavage directed by the endogenous let-7-programmed RISC in the HeLa S100 extract and by the RISC programmed with exogenous let-7 siRNA duplex in Drosophila embryo lysate . This finding suggests that single-stranded 2′-O-methyl oligonucleotides are not efficiently transmitted to the progeny of injected animals. To circumvent this problem, we next injected 2′-O-methyl oligonucleotides directly into larvae and examined the phenotypes of the injected animals. The lin-4 miRNA functions in L1/L2 larvae, and we have found that, in our hands, L1 larvae do not survive microinjection (data not shown); thus, it was not possible to assay for inhibition of lin-4 function by direct injection. In contrast, let-7 functions during the L4 stage, and we found that L2 and L3 larvae survive the microinjection procedure  strain and compared the penetrance of the phenotypes with those observed for injection into wild-type. Consistent with the idea that the injected oligonucleotide specifically inactivates let-7, the absence of alae- and vulval-bursting phenotypes were both suppressed in the lin-41(ma104) mutant strain (lin-41(ma104) strain). The observed suppression (64%) was nearly identical to that reported for a let-7, lin-41 genetic double mutant  of the miRNAs lin-4 and let-7  is less than the amount of siRNA transfected. These data suggest that only a fraction of the transfected siRNA forms RISC. Furthermore, the data are consistent with stoichiometric and irreversible binding of the 2′-O-methyl oligonucleotide to RISC in vivo.Our in vitro and in vivo studies using 2′-O-methyl oligonucleotide by RISC and, by inference, recognition of target RNA by RISC are qualitatively different from the simple binding of two complementary nucleic acids by basepairing. We observed that RISC function was far more readily inhibited by binding a 2′-O-methyl oligonucleotide to RISC than by binding the same 2′-O-methyl oligonucleotide to the site of RISC recognition on a target RNA. A clear implication of this finding is that RISC does not acquire its RNA target by a passive basepairing mechanism that zippers together 21 nt of complementary RNA. Thus, RNAi is not merely a form of antisense inhibition in which the antisense strand is stabilized in a duplex. Rather, an active mechanism—perhaps involving target sequences flanking the region of complementarity—underlies the specificity and efficiency of RISC targeting.Our data hint that recognition of the 2′--O-methyl oligonucleotides to probe miRNA function in vivo. Injection of a 2′-O-methyl oligonucleotide complementary to the let-7 miRNA into C. elegans larvae phenocopied a let-7 loss-of-function mutation, demonstrating that 2′-O-methyl oligonucleotides can disrupt the function of a single miRNA in vivo. These data, combined with our studies in vitro and in cultured cells, show the promise of 2′-O-methyl oligonucleotides as a tool for dissecting the function of the numerous miRNAs found in a wide range of organisms. In this regard, 2′-O-methyl oligonucleotides provide a tool similar in practice, but mechanistically distinct from, RNAi itself and thus may facilitate the study of small RNA function in cases in which classical genetic mutations in miRNA genes are unavailable.Finally, we have shown the utility of 2′Drosophila embryo lysate preparation, in vitro RNAi reactions, and cap-labeling of target RNAs were as described elsewhere  were 5′-CAU CAC GUA CGC GGA AUA CUU CGA AAU GUC C-3′ and 5′-Bio-CAU CAC GUA CGC GGA AUA CUU CGA AAU GUC C-3′ (complementary to the Pp-luc siRNA sense strand); 5′-GGA CAU UUC GAA GUA UUC CGC GUA CGU GAU G-3′ and 5′-Bio-A CAU UUC GAA GUA UUC CGC GUA CGU GAU GUU-3′ (complementary to the Pp-luc antisense strand); and 5′-Bio-UCU UCA CUA UAC AAC CUA CUA CCU CAA CCU U-3′ (complementary to let-7); 5′ biotin was attached via a six-carbon spacer arm.Synthetic siRNA (Dharmacon, Lafayette, Colorado, United States) was deprotected according to the manufacturer, annealed b, 2001c,O-methyl oligonucleotide (10 pmol) was incubated for 1 h on ice in lysis buffer containing 2 mM DTT with 50 μl of Dynabeads M280  to immobilize the oligonucleotide on the beads. To ensure that the tethered oligonucleotide remained in excess when more than 50 nM siRNA was used, 20 pmol of biotinylated 2′-O-methyl oligonucleotide was immobilized. For RISC capture assays, siRNA was preincubated in a standard 50 μl in vitro RNAi reaction for 15 min at 25°C. Then, the immobilized 2′-O-methyl oligonucleotide was added to the reaction and incubation continued for 1 h at 25°C. After incubation, beads were collected using a magnetic stand . The unbound supernatant was recovered and an aliquot assayed for RISC activity as previously described  using Lipofectamine 2000  according to the manufacturer's protocol, first with various concentrations of siRNA targeting Pp-luc mRNA. After 6 h, the cells were washed with PBS and the media replaced. On the next day, the cells were cotransfected with Rr-luc-expressing (0.1 μg/well) and Pp-luc-expressing (0.25 μg/well) plasmids and 2′-O-methyl oligonucleotides using Lipofectamine 2000 (GIBCO) according to the manufacturer's protocol. The luciferase activity was measured 24 h later with the Dual Luciferase assay kit (Promega, Madison, Wisconsin, United States) using a Mediators Diagnostika (Vienna, Austria) PhL luminometer.HeLa S3 cells were transfected in a 24-well plate (200 mmlet-7 function, 1 mg/ml let-7-complementary 2′-O-methyl oligonucleotide in water (100 μM) was injected into either wild-type (N2) or lin-41(ma104) L2 larvae. Injection of L2 larvae was essentially as described elsewhere  and Mini Complete Protease Inhibitor cocktail (1 tablet/10 ml solution) (Roche, Basel, Switzerland) using a stainless-steel Dounce homogenizer (Wheaton Incorporated, Millville, New Jersey, United States). The homogenized extract was clarified by a centrifugation at 13,817 × let-7 miRNA, the beads were boiled for 10 min in 20 μl of SDS loading buffer (10 mM Tris–HCl [pH 6.8], 2% [w/v] SDS, 100 mM DTT, and 10% [v/v] glycerol). Proteins were resolved by SDS-PAGE on an 8% gel and transferred to Hybond-C membrane . To detect GFP-tagged ALG-1, ALG-2, and RDE-4 proteins, the membrane was incubated overnight at 4°C with either monoclonal anti-GFP (Roche) or an affinity-purified polyclonal anti-RDE-4 antibody (To recover the proteins associated with the antibody  diluted Immunoprecipitation of GFP-tagged ALG-1/ALG-2 complexes was performed by preclearing worm extract with 50 μl of protein G–agarose beads (Roche) per 5 mg of total protein for 1 h at 4°C. The cleared extract was then incubated with 10 μg of monoclonal antibody anti-AFP 3E6  for 1 h at 4°C followed by 50 μl of protein G–agarose. The agarose beads were then washed three times with ice-cold homogenization buffer.let-7 miRNA was monitored by Northern blotting. RNA was eluted from the immobilized 2′-O-methyl oligonucleotide by digestion with 1 mg/ml proteinase K in 200 mM Tris–HCl (pH 7.5), 25 mM EDTA, 300 mM NaCl, 2% (w/v) SDS at 50°C for 30 min, followed by extraction with phenol–chloroform, and recovered by precipitation with ethanol. Recovered RNA was resuspended in 10 μl of formamide-loading buffer (98% [v/v] deionized formamide, 10 mM EDTA, 0.025% [w/v] xylene cyanol, 0.025 % [w/v] bromophenol blue), heated to 100°C for 2 min. RNA was resolved on a 15% denaturing polyacrylamide gel, transferred to Hybond-N membrane (Amersham Biosciences), and detected by Northern blot analysis using a 5′-32P-radiolabeled antisense let-7 RNA probe (UAU ACA ACC UAC UAC CUC AUU) as described elsewhere (Depletion of lsewhere .http://www.ncbi.nlm.nih.gov/Genbank/) accession number for Photinus pyralis is X65324 and for Renilla reniformis is AF025846.The GenBank (http://www.sanger.ac.uk/Software/Rfam/index.shtml) accession numbers for the let-7 family members are MI0000060–MI0000068, MI0000433, and MI0000434.Rfam (http://www.ncbi.nlm.nih.gov/LocusLink/) ID numbers for the genes discussed in this paper are alg-1 (181504), alg-2 (173468), bantam (117376), let-7 (266954), lin-4 (266860), lin-41 (172760), miR-14 (170868), and rde-4 (176438).The LocusLink (


Batrachochytrium dendrobatidis has been implicated in the decline and extinction of numerous frog species worldwide. In Queensland, Australia, it has been proposed as the cause of the decline or apparent extinction of at least 14 high-elevation rainforest frog species. One of these, Taudactylus eungellensis, disappeared from rainforest streams in Eungella National Park in 1985–1986, but a few remnant populations were subsequently discovered. Here, we report the analysis of B. dendrobatidis infections in toe tips of T. eungellensis and sympatric species collected in a mark-recapture study between 1994 and 1998. This longitudinal study of the fungus in individually marked frogs sheds new light on the effect of this threatening infectious process in field, as distinct from laboratory, conditions. We found a seasonal peak of infection in the cooler months, with no evidence of interannual variation. The overall prevalence of infection was 18% in T. eungellensis and 28% in Litoria wilcoxii/jungguy, a sympatric frog that appeared not to decline in 1985–1986. No infection was found in any of the other sympatric species. Most importantly, we found no consistent evidence of lower survival in T. eungellensis that were infected at the time of first capture, compared with uninfected individuals. These results refute the hypothesis that remnant populations of T. eungellensis recovered after a B. dendrobatidis epidemic because the pathogen had disappeared. They show that populations of T. eungellensis now persist with stable, endemic infections of B. dendrobatidis.The chytrid fungus  Recapture experiments provide evidence that some amphibian species can now persist with infections of the pathogenic chytrid fungus and suggests, for example, that frogs and fungus might be coevolving Lauranctil 1998  and was til 1998 . B. denddeclines , althougdeclines  had direB. dendrobatidis infection on toe tips collected between 1994 and 1998 from six species of frogs at Eungella National Park in east-central Queensland, Australia ((Rheobatrachus vitellinus) disappeared suddenly from relatively undisturbed rainforest streams, and it is now considered to be extinct ((Taudactylus eungellensis) also disappeared, but it was later found to have persisted in a few small populations  of the 474 toes assessed. Four species showed no sign of infection, while we detected infections on 58 (18.4%) of 316 toes of T. eungellensis and 13 (34%) of 47 toes of frogs identified at the time as Litoria lesueuri . The most parsimonious model (i.e., one that minimizes the Akaike Information Criterion [AIC]) included each of these predictors but no interaction terms.Using a logistic model with season (summer, autumn, winter, and spring), species, and site as predictor variables, we found significant effects of each variable on infection, corrected for the effects of the other variables . A significantly smaller proportion was infected at Rawson Creek (10.5%) than at Dooloomai Falls (26.7%) or Tree Fern Creek (25.0%). There was a marginally significant difference overall in the infection levels among males, females, and subadults (Δdev = 5.9, df = 2, p = 0.052). However, when site was included in the model, any suggestion of a difference in infection level between males, females, or subadults disappeared. When we separated the main T. eungellensis populations, it was apparent that the difference in prevalence between those categories was influenced by the population at Tree Fern Creek, where 46% of males (n = 13) and no females or subadults were infected  differeinfected . When weT. eungellensis varied significantly among seasons (Δdev = 14.605, df = 3, p = 0.002), but not among years (Δdev = 3.433, df = 3, p = 0.26). Further, there was no evidence that the pattern of seasonal variation in infection changed among years (Δdev = 5.561, df = 6, p = 0.49). Infection was most prevalent (37.8%) during the winter months (1 June to 31 August) and least prevalent (11.3%) during the summer months (1 December to 28/29 February). Comparing the two sites with the largest sample sizes, Dooloomai Falls and Rawson Creek, there was no evidence that they had differing seasonal patterns of infection (Δdev = 5.32, df = 3, p = 0.150), although the level of infection overall was much higher at Dooloomai Falls (log odds ratio = 1.1612, standard error [se] = 0.3586, p = 0.0012). Infection levels were much higher in winter and spring combined than in summer and autumn (log odds ratio = 1.360, se = 0.3589, p = 0.00015), and there was no evidence of infection levels differing between winter and spring or between summer and autumn (Δdev = 1.107, df = 2, p = 0.5). The seasonal changes in infection at the two sites are shown in At these three sites, levels of infection among B. dendrobatidis was detected in 13 of the 47 L. wilcoxii/jungguy examined, giving an estimated prevalence of 27.7%, with a 95% confidence interval ranging from 15.6% to 42.6%. There was no evidence that prevalence of infection differed among males, females, and subadults (Δdev = 2.32, df = 2, p = 0.31); among sites (Δdev = 0.175, df = 2, p = 0.91); or among seasons (Δdev = 1.44, df = 1, p = 0.7) (each of the above Δdev terms is corrected for the other terms in the model).L. wilcoxii/jungguy and T. eungellensis was hampered by the fact that prevalence of infection on T. eungellensis differed between sites, and that the sampled populations of the two frog species had largely disjunct distributions. When the data were pooled over all sites, there was insufficient evidence to reject a null hypothesis of equal prevalence in the two species, whether the effects of season were allowed for (Δdev = 0.15, df = 1, p = 0.7) or not (Δdev = 2.09, df = 1, p = 0.15).Comparing the prevalence of infection between L. wilcoxii/jungguy and T. eungellensis recaptured at any stage later in the study, grouped by their infection status at first capture. A logistic model with recapture as the response and species and infection status as predictors produced no evidence that the effect of B. dendrobatidis infection on recapture probability differed between the species (Δdev = 0.002, df = 1, p = 0.96). In both species, the probability of recapture was significantly lower for infected frogs than for uninfected frogs , correcting for the substantially higher overall recapture rate of T. eungellensis relative to L. wilcoxii/jungguy.B. dendrobatidis may affect survival, but confounds the probability of recapturing a frog that is present at a site with the probability that a frog is still present at the site. The more sophisticated analysis that follows separates these two components, although it cannot distinguish between death and permanent emigration from the site. For brevity, we refer to continued presence at the site as “survival.”This simple analysis suggests that Investigating mark–recapture data for both Rawson Creek and Dooloomai Falls, we found that the best model (on the basis of minimizing the AIC) for which there were sufficient data to estimate almost all parameters had the probability of recapture varying with time but not group , and the survival probability differing between the groups but constant through time. However, further analysis in both cases showed that there was no evidence that survival differed between infected and uninfected frogs: At both sites, the group effect was due to survival being highest in females, intermediate in males, and lowest in subadults ; Table 4T. eungellensis, a rainforest frog that almost disappeared as a result of major die-offs in the Eungella area in the mid 1980s, now persist with stable infections of B. dendrobatidis. This does not imply that this pathogen cannot have been responsible for the decline. One hypothesis that can be discarded, however, is that B. dendrobatidis drove frog populations to low levels, consequently became locally extinct itself, and that frog populations subsequently recovered or stabilised in the absence of the pathogen. Two other hypotheses are consistent with our observed results.Our results show unequivocally that remnant populations of B. dendrobatidis was not responsible for the initial decline of T. eungellensis populations. These populations declined more than a decade before this chytrid was formally identified, and the declines occurred over a very short period , our results show that infection with B. dendrobatidis did not inevitably lead to rapid death in T. eungellensis.Our mark–recapture analysis did not find consistent evidence that captured . TogetheL. wilcoxii/jungguy appears not to have declined at the same time as T. eungellensis and R. vitellinus and, in our data, it has a high prevalence of infection. It therefore is a candidate reservoir host. Whether the prevalence of B. dendrobatidis differs between the sibling species L. wilcoxii and L. jungguy at sites where they are sympatric would be an interesting question, but cannot be answered from our study. The L. lesueuri complex has a widespread distribution throughout streams on the east coast of Australia  were histologically prepared in transverse sections stained with Ehrlich's haematoxylin for skeletochronological assessment . For both mark–recapture and analysis of prevalence, we grouped the data into 3-mo austral seasons . Mark–recapture modelling of survival requires that several assumptions be satisfied. Most critically, every marked individual present in the population at a given time has the same probability of recapture as all other members of its group, and the same probability of surviving to the next time interval. We used a parametric bootstrap (With the software MARK , we usedootstrap  to test ootstrap . Since t