bluuebunny/crossref_metadata_2025 huggingface dataset:
A simple method for the two‐step preparation of two pure haemorphins from a total haemoglobin peptic hydrolysate by conventional low‐pressure chromatographies
Authors: Choisnard Luc , Durand David , Vercaigne‐Marko Dominique , Nedjar‐Arroume Naima , Dhulster Pascal , Guillochon Didier
Published: November 2003

The development of a simple purification method for two haemorphins, VV‐haemorphin‐7 (Val‐Val‐Tyr‐Pro‐Trp‐Thr‐Gln‐Arg‐Phe) and VV‐haemorphin‐4 (Val‐Val‐Tyr‐Pro‐Trp‐Thr), from a total peptic haemoglobin hydrolysate is described. Cation‐exchange chromatography on CM‐Sephadex was used as a pre‐fractionation step for the hydrolysate. VV‐haemorphin‐7 and VV‐haemorphin‐4 were eluted in two different fractions. The second and final purification step was hydrophobic‐interaction chromatography on phenyl‐Sepharose, which allowed the isolation of the two pure haemorphins. Haemoglobin and globin hydrolysates were compared as starting materials. For easy recovery of haemorphins and easy adjustment of conditions for final purification, a volatile buffer, ammonium acetate buffer, pH 6.5, was employed. This process, which allowed the preparation of pure haemorphins from a total protein hydrolysate, could be scaled up and used in the food industry.
Production of non‐alcoholic beer using free and immobilized cells of Saccharomyces cerevisiae deficient in the tricarboxylic acid cycle
Authors: Navrátil Marián , Dömény Zoltán , Šturdík Ernest , Šmogrovičová Daniela , Gemeiner Peter
Published: November 2003

Production of non‐alcoholic beer using Saccharomyces cerevisiae has been studied. Non‐recombinant mutant strains with a defect in the synthesis of tricarboxylic‐acid‐cycle enzymes were used and applied in both free and pectate‐immobilized form, using both batch and packed‐bed continuous systems. After fermentation, basic parameters of the beer produced by five mutant strains were compared with a standard strain of brewing yeast. Results showed that the beer prepared by mutant yeast cells was characterized by lower levels of total alcohols, with ethanol concentrations between 0.07 and 0.31% (w/w). The organic acids produced, especially lactic acid, in concentrations up to 1.38 g·l−1 had a strong protective effect on the microbial stability of the final product and thus the usual addition of lactic acid could be omitted. Application of the yeast mutants appears to be a good alternative to the classical methods for the production of non‐alcoholic beer.
Approach to assay validation for the development of biopharmaceuticals
Authors: Lundblad Roger L.
Published: November 2003

The development, manufacture and characterization of biopharmaceuticals requires the use of a variety of analytical procedures. Each assay must be validated to demonstrate that it is suitable for its intended purpose, either as an in‐process analysis for the characterization of critical product/process attributes or final product characteristics. This paper is a brief review of the various considerations required for the validation of an assay procedure for use within Good Laboratory Practices (‘GLP’) or current Good Manufacturing Practices (‘cGMP’).
Changes in the overall extent of protein glycosylation by Chinese hamster ovary cells over the course of batch culture
Authors: Yuk Inn H. Y. , Wang Daniel I. C.
Published: November 2003

Glycosylation of recombinant proteins can change during the culture of animal cells. Since lipid‐linked oligosaccharides (LLOs) are the carbohydrate donors in N‐linked glycosylation, their availability is postulated to influence the extent of glycosylation. To test this hypothesis, relative LLO and glycosylation levels in Chinese hamster ovary (CHO) cells were monitored over the course of batch culture for corresponding changes. Radiolabelling studies reveal that throughout the length of culture, intracellular LLO levels remained within a 2‐fold range and overall CHO protein glycosylation varied by less than 30%. The implications of these results and hypotheses to explain the findings are discussed.
A high‐stringency proteomics concept aimed for generation of antibodies specific for cDNA‐encoded proteins
Authors: Gräslund Susanne , Falk Ronny , Brundell Eva , Höög Christer , Ståhl Stefan
Published: November 2003

A novel dual bacterial expression system, designed for high‐throughput generation of antibodies specific for cDNA‐encoded proteins, is presented. The concept involves parallel expression of cDNA‐encoded proteins, in two vector systems, as fusions with two different tags, both enabling single‐step affinity purification under denaturing conditions. One of the fusion tags includes a portion with documented immunopotentiating effect to stimulate antibody production, and the generated fusion proteins are used to elicit antibodies. The second fusion protein is used in an immobilized form as an affinity ligand to enrich, from the generated antisera, antibodies with selective reactivity to the cDNA‐encoded part. To evaluate the system, five cDNA clones from a mouse testis cDNA library were expressed, and antibodies to these cDNA‐encoded proteins were generated, enriched and used in blotting procedures to determine expression patterns for the native proteins corresponding to the cDNAs. The five antibody preparations showed specific reactivity to the corresponding recombinant cDNA‐encoded proteins, and three of the five antibodies gave specific staining in Western‐blot screening of various cell types and tissue homogenates. When the same five cDNAs were processed and analysed using a single‐vector method, antibodies with a more non‐specific staining were generated. We thus conclude that the presented dual‐vector method offers a highly stringent strategy for generation of monospecific polyclonal antibodies.
A new method for the preparation of human parathyroid hormone 1–34 peptides
Authors: Xiu Zhaoyang , Li Min , Zhou Suijing , Dou Hong , Zhou Heyue , Chen Changqing
Published: November 2003

An engineered Escherichia coli strain, BL21 (DE3)/pGEX‐4T‐human parathyroid hormone (hPTH) (1–34), was constructed by oligonucleotide annealing and PCR amplification of the target gene, and then by ligating it with the pGEX‐4T‐3 vector and transferring into the BL21 host. The soluble glutathione S‐transferase (GST) fusion protein GST‐hPTH (1–34), expressed from BL21 (DE3)/pGEX‐4T‐hPTH (1–34), was harvested after fermentation and purification by affinity chromatography. Following double cleavage by thrombin and prolyl endopeptidase, about 0.6 g/l intact hPTH (1–34) was harvested. The product was checked by HPLC MS and N‐terminal sequence analysis. The purified recombinant hPTH (1–34) stimulates adenylate cyclase in rabbit renal cortical cell membranes to exactly the same extent as synthetic hPTH standards, indicating that the recombinant product has full biological activity.
Application of statistically based experimental designs for the optimization of exo‐polysaccharide production by Cordyceps militaris NG3
Authors: Xu Chun‐Ping , Kim Sang‐Woo , Hwang Hye‐Jin , Yun Jong‐Won
Published: November 2003

Statistically based experimental designs were applied to the optimization of medium composition for exo‐polysaccharide production by Cordyceps militaris NG3 in shake‐flask cultures. First, the Plackett–Burman design was used to search for the main factors on mycelia and exo‐polysaccharide production. Among these variables, sucrose and corn steep powder were found to be two significant factors and had positive effects on mycelial yield (with confidence level >80%) and exo‐polysaccharide production (with confidence level >90%). Subsequently, to study the mutual interactions between variables, the effects of the two main factors on exo‐polysaccharide production were further investigated using a central composite design. The optimal composition was found to be 1.03 g/l corn steep powder, 2.95 g/l sucrose, 0.1 g/l K2HPO4, 0.5 g/l MgSO4·5H2O and 0.1 g/l KNO3 for the enhanced production of the exo‐polysaccharide, which was 2.604 g/l in shake‐flask cultures. Under optimal culture conditions, the maximum exo‐polysaccharide concentration in a 5 l stirred‐tank bioreactor was 3.8 g/l.
An analysis of target preferences of Escherichia coli outer‐membrane endoprotease OmpT for use in therapeutic peptide production: efficient cleavage of substrates with basic amino acids at the P4 and P6 positions
Authors: Okuno Kazuaki , Yabuta Masayuki , Ohsuye Kazuhiro , Ooi Toshihiko , Kinoshita Shinichi
Published: November 2003

The Escherichia coli outer‐membrane endoprotease OmpT mainly cleaves peptide bonds between consecutive basic amino acids. The effect of adjacent residues on cleavage efficiency is currently unknown, except at positions P2 and P2′. Therefore we investigated the effects of amino acid residues upstream of the cleavage site on the ability of OmpT to cleave efficiently a fusion protein carrying human glucagon‐like peptide‐1 (7–37) in 4 M urea. The P1–P10 residues were replaced by Ala and each substrate was subjected to OmpT digestion. The replacement of Arg residue at P1 blocked the cleavage due to the loss of the cleavage site, and the replacement of Arg residue at P4 maximally reduced the cleavage rate. Conversely, cleavage efficiency increased on replacing Glu at P6. Substitution of the residues at P4 and P6 with several different amino acids showed that OmpT preferred basic residues at these positions, whereas acidic residues had a negative effect. This was also shown to be true with synthetic decapeptide substrates in the absence of urea. The kcat/Km ratio increased with basic residues at P4 or P6, mainly due to a lower Km rather than an increase in kcat. On the basis of these findings, we prepared a fusion protein carrying human atrial natriuretic peptide (ANP), a drug for acute congestive heart failure. OmpT released mature ANP from the E. coli‐expressed fusion protein. As expected, the introduction of an Arg residue at P4 and P6 enhanced the release of ANP.
Guide DNA technique in bacterial ribonuclease P reaction for effective processing of tRNA precursor
Authors: Tanaka Terumichi , Hori Yoshiaki , Kikuchi Yo
Published: November 2003

Previously, we found that a small (approx. 20‐mer) DNA hybridizing to the 5′‐leader region of a tRNA precursor enhances the cleavage efficiency in bacterial ribonuclease P reaction. We named this technique the ‘guide DNA technique’. Detailed analyses showed that the length of the guide DNA, concentration of the guide DNA and the hybridizing position affected the cleavage efficiency: for an effective cleavage reaction, guide DNA should be designed to hybridize to the region on the cleavage site, should be 20 bases or more in length and should be of high concentration. The presence of a 5′‐flanking region in the DNA did not affect the cleavage reaction. The guide DNA technique is a useful tool for effective preparation of mature tRNA molecules in vitro.
Chemokine receptors on infiltrating leucocytes in inflammatory pathologies of the central nervous system (CNS)
Authors: Trebst C. , Staugaitis S. M. , Tucky B. , Wei T. , Suzuki K. , Aldape K. D. , Pardo C. A. , Troncoso J. , Lassmann H. , Ransohoff R. M.
Published: November 2003

Haematogenous leucocytes enter the central nervous system (CNS) during diverse disorders of varied aetiologies. Understanding the trafficking cues that mediate CNS leucocyte infiltration might promote the development of flexible and selective means to modulate inflammation to achieve clinical benefit. The trafficking machinery of leucocytes has been elucidated during the past decade and consists of cell‐surface adhesion molecules, chemoattractant cytokines (chemokines) and their receptors. Recent work in our laboratory characterized chemokine receptors found on T lymphocytes and monocytes in brain sections from subjects with one pathological subtype of multiple sclerosis (MS), an immune‐mediated inflammatory demyelinating disease. In these tissues, the types 1 and 5 CC chemokine receptors (CCR1 and CCR5) were detected on perivascular monocytic cells whereas only CCR5 was present on parenchymal macrophages. The type 3 CXC chemokine receptor (CXCR3) was present on virtually all CD3‐positive T cells. In the current study, we evaluated the expression of these receptors on the infiltrating cells present in cases of other inflammatory CNS disorders including those of dysimmune, infectious, neoplastic, and vascular aetiology. Perivascular and parenchymal monocytic cells expressed CCR1 in all cases and CXCR3 was consistently present on a substantial proportion of CD3+ T cells. The occurrence of CCR5 on parenchymal macrophages was much less uniform across the varied disorders. These data implicate CCR1 in monocyte infiltration of the CNS and are consistent with reports of studies in CCR1‐deficient mice. CXCR3 is also likely to play a role in accumulation  of  T  cells  in  the inflamed  CNS.  By  contrast, our findings suggest that regulation of CCR5 on phagocytic macrophages may be contingent on the lesion environment.
Role of buffalo in the socioeconomic development of rural Asia: Current status and future prospectus
Authors: NANDA Amarjit Singh , NAKAO Toshihiko
Published: November 2003

Buffalo has been an integral part of livestock agriculture in Asia for over 5000 years producing draft power, milk, meat and hides. Even today, 153 million buffalo provide 76 734 759 t of milk and 3 089 875 t of meat, and in several countries, up to 30% of the draft power for agricultural operations. Unfortunately, however, the species did not receive the attention of the policy makers and the researchers in accordance with its merits, which resulted in buffalo population decline in several eastern Asian countries. The present review highlights the importance of buffalo to the economic and social fabric, especially of the small and landless rural poor, of several countries in Asia. Some major national and international efforts being made to improve buffalo production have also been cited.
Amino acid nutrition in egg production industry
Authors: ISHIBASHI Teru , YONEMOCHI Chisato
Published: November 2003

The egg production industry is facing various problems that need to be solved. For amino acid nutrition to achieve scientific and economical feeding of laying hens, it is necessary to elucidate the content, digestibility, or availability of nutrients of feedstuffs and feeds and the requirement of amino acids for laying hens. In addition, improvement to quality of eggs and meat of spent hens, methods of management and development of new feedstuffs are essential. For sustainable animal production, decrease in excreta and animal welfare should be studied. The real‐time determination of content and digestibility of amino acids in feedstuffs are essential for formation of feeds. Recent advances in the near infrared reflectance analysis will be able to determine the content, digestibility and availability of nutrients in feedstuffs and feeds, if we have a supplemental amount of conventional analysis to define the calibration population. The amino acid requirements are affected by various factors. Therefore the method to quickly and exactly determine amino acid requirements in response to various factors is necessary. By using plasma free amino acid concentration as a criterion, it is possible to determine amino acid requirements in various conditions of laying hens within a short experimental period, repeatedly using the same animals. Because the amino acid requirements differ among individual animals, it should be expressed as grams per hen per day. Practically, it is impossible to formulate various feeds for individual hens. The various expressions have been developed and these expressions have advantages and disadvantages. The nitrogen excretion of laying hens is easily reduced by reducing dietary nitrogen levels and restricting the feed intake. The availability of amino acid may be improved by feeding management, and supplementing enzyme, but the quality of eggs and meat of spent hens and welfare of laying hens are not affected by amino acid nutrition.
Growth hormone treatment affects brain neurotransmitters and thyroxine
Authors: Burman P. , Hetta J. , Wide L. , Månsson J‐E. , Ekman R. , Karlsson F. A.
Published: November 2003

OBJECTIVE Binding sites specific for growth hormone have been identified in the brain, but the action of GH on the central nervous system is still poorly understood.

DESIGN In a double‐blind, placebo‐controlled 21‐month trial with a cross‐over design, with each treatment period lasting for 9 months, we investigated the long‐term effect of GH on the cerebrospinal fluid (CSF) concentrations of some brain neurotransmitters and thyroid hormones of importance for mood and cognition.

PATIENTS Twenty‐four patients with documented GH deficiency acquired in adult life took part.

RESULTS Analysis of CSF collected at the end of the two treatment periods showed that the GH concentration was related to the administered dose of rhGH (r = 0.56, P = 0.0044). After rhGH treatment the concentration of the dopamine metabolite homovanillic acid (HVA) had decreased from 218 ± 80 to 193 ± 82 nmol/l (P = 0.002) and that of the excitatory acid aspartate had increased from 233 ± 81 to 313 ± 116 nmol/l (P = 0.032). No effects were observed on the concentrations of 5‐hydroxyindole‐acetic acid (the serotonin metabolite) and of 3‐methoxy‐4‐hydroxyphenyl glycol (the noradrenaline metabolite), or on those of glutamate, glycine and β‐endorphin. However, both CSF and serum levels of free T4 decreased, from 19.8 ± 6.1 to 16.6± 5.7 nmol/l (P = 0.0002) and 17.0 ± 5.0 to 13.7 ± 4.3 nmol/l (P = 0.0001), respectively. The concentration of total T3 was not measurable in CSF but increased in serum from 1.41 to 1.53 nmol/l (P = 0.01).

CONCLUSION The study demonstrates a passage of GH from the circulation into the CSF. The observed changes in homovanillic acid and free T4 are similar to those reported after successful treatment of depressive disorders with antidepressant drugs, and may reflect a beneficial effect of GH on mood and behaviour.
Characterization of inhibin immunoreactivity in post‐menopausal women with ovarian tumours
Authors: Burger H. G. , Robertson D. M. , Cahir N. , Mamers P. , Healy D. L. , Jobling T. , Groome N.
Published: November 2003

BACKGROUND AND OBJECTIVE We have previously reported elevated serum immunoreactive inhibin (INH) levels in patients with ovarian malignancies, particularly granulosa cell and mucinous tumours. The present study was designed to compare INH measurements using a heterologous radioimmunoassay with cross‐reactivity for inhibin α‐subunit derived peptides with measurements obtained using a new ELISA specific for dimeric inhibin‐A. It was hypothesized that granulosa cell tumours may secrete significant quantities of inhibin‐A whereas mucinous tumours were unlikely to do so because of the lack of a relation between INH and FSH measurements in the latter group.

DESIGN Serum samples obtained from women found to have ovarian cancer were assayed using the heterologous radioimmunoassay (the Monash assay) and using an ELISA specific for dimeric inhibin (the Groome assay) and the results were compared.

PATIENTS Samples for assay were available from 69 normal post‐menopausal control women, 12 patients with mucinous tumours of the ovary, 26 with serous tumours, 7 with granulosa cell tumours and 8 with various other ovarian tumours. Patients were post‐menopausal or had been subjected to bilateral oophorectomy at the time these samples were collected.

MEASUREMENTS The Monash and Groome assays were carried out as described previously. The upper limit of normal for post‐menopausal women in the Monash assay was 122 U/l and for the Groome assay was calculated to be 32 ng/l.

RESULTS Among the 69 normal subjects, 4 were found to have elevated inhibin levels using the Monash RIA (133–190 U/l) and 4 were found to have elevated levels in the Groome ELISA (45.5–55.3 ng/l). Among 12 patients with mucinous tumours, 10 (83%) had elevated inhibin levels using the Monash assay but only 3 (25%) had elevated levels with the Groome assay (P < 0.005). Among 26 with serous tumours, 15 (58%) had elevated levels in the Monash assay but only 1 (4%) in the Groome assay (P < 0.001). Among 7 samples from patients with granulosa cell tumours, 100% were elevated in the Monash assay and 71% in the Groome assay (NS, non‐significant). In a miscellaneous group of tumours all 8 had elevated levels in the Monash assay and 2 in the Groome assay (P < 0.001).

CONCLUSIONS It was concluded that in normal post‐menopausal subjects, INH is generally undetectable or present at low levels, consistent with the loss of ovarian function. The majority of granulosa cell tumours appear to secrete significant amounts of dimeric inhibin‐A, whereas mucinous tumours secrete predominantly other forms of INH, presumably related to the α‐subunit. Serous tumours may also secrete inhibin‐related peptides but not dimeric inhibin‐A. The nature of the inhibin related peptides produced by epithelial ovarian cancers remains to be characterized.
A case of silent 21‐hydroxylase deficiency with persistent adrenal insufficiency after removal of an adrenal incidentaloma
Authors: Nagasaka Shoichiro , Kubota Ken , Motegi Takashi , Hayashi Eiji , Ohta Manabu , Takahashi Keiichi , Takahashi Takashi , Iwasaki Yoshiaki , Koike Morio , Nishikawa Tetsuo , Sasano Hironobu , Murakami Toru
Published: November 2003

A case of an adrenal incidentaloma in a 57‐year‐old man with silent 21‐hydroxylase deficiency is reported. Abdominal computed tomography revealed a right adrenal tumour of 6cm in diameter. There was no evidence of adrenal hormone excess. However, after surgical removal of the adrenal tumour, the patient developed acute adrenal insufficiency. Adrenocortical function has remained low ever since surgery. Pathological examination of the tumour revealed a cortical adenoma. On the basis of increased plasma renin activity and serum 17α‐hydroxyprogesterone concentration and decreased 21‐hydroxylase activity of the adenoma tissue, the patient was diagnosed as having systemic 21‐hydroxylase deficiency. There are two possible mechanisms for the persistent adrenal insufficiency; first the residual left adrenal gland may have been originally hypo‐functioning for some reason and, second, the left gland may have lost the ability to regenerate following prolonged suppression by the adenoma. In cases of adrenal incidentalomas with 21‐hydroxylase deficiency, the indications for surgical removal should be carefully considered.
Pituitary apoplexy after leuprolide administration for carcinoma of the prostate
Authors: Morsi Amr , Jamal Sophie , Silverberg Jay D. H.
Published: November 2003

The syndrome of pituitary apoplexy has been reported to occur after the administration of several different medications. We report a case in which pituitary apoplexy developed shortly after the administration of leuprolide in a patient with prostate cancer. Leuprolide is a potent gonadotrophin releasing hormone (GnRH) analogue used to suppress leuteotrophic hormone (LH) and testosterone levels in patients with metastatic prostate cancer. LH and testosterone levels actually rise in the first week after its administration before becoming suppressed. We suspect that this acute stimulating effect of leuprolide is linked to the acute onset of pituitary apoplexy in a patient with a possible gonadotrophoma.
Polymorphism of glycogen synthetase gene in polycystic ovary syndrome
Authors: Rajkhowa M. , Talbot J. A. , Jones P. W. , Clayton R. N.
Published: November 2003

OBJECTIVE Polycystic ovary syndrome is a heterogeneous disorder associated with a moderate degree of insulin resistance and a higher risk of developing NIDDM. The exact mechanism of insulin resistance is unclear. This study examines the frequency of an Xbal polymorphism of the glycogen synthetase gene (A2 allele) as a marker of insulin resistance and seeks to relate the presence of the A2 allele to indices of insulin sensitivity in women with polycystic ovary syndrome (PCOS).

METHODS Insulin sensitivity was assessed by fasting insulin measurements, as well as following oral glucose tolerance test. An i.v. insulin tolerance test was performed to measure the rate of endogenous blood glucose disposal following an i.v. bolus of insulin. Restriction fragment length polymorphism was performed with Xbal digestion of PCR amplified product to detect the presence of A1 and A2 allele.

PATIENTS Seventy‐one obese (BMI > 25.1) and 19 non‐obese (BMI < 25) women with PCOS, and 62 controls (33 obese and 29 non‐obese) participated in the study.

RESULTS Obese PCOS had significantly higher fasting insulin ( P = 0.002) compared to obese controls. There was no difference between non‐obese PCOS and controls. Twenty per cent of obese PCOS had impaired glucose tolerance.

The A1A2 genotype was detected in 16 of the 150 (10.7%) subjects examined. Of these, 11/88 (12.5%) were PCOS and 5/62 (8%) were controls. The A2A2 genotype was not present in any of the subjects. The A1A2 genotype was not detected in any of the subjects with impaired glucose tolerance. There was no significant difference in the incidence of the A1A2 genotype between PCOS and controls or between the individual groups. There was no association between the presence of the A1A2 genotype and indices of insulin sensitivity.

CONCLUSION The Xbal polymorphism (A2 allele) of the glycogen synthetase gene was not over represented in the PCOS subject and did not relate to the indices of insulin sensitivity or glucose intolerance.
Somatic mutations in the RET proto‐oncogene in sporadic medullary thyroid carcinoma
Authors: Marsh Debbie J. , Learoyd Diana L. , Andrew Scott D. , Krishnan Lalitha , Pojer Ruth , Richardson Anne‐Louise , Delbridge Leigh , Eng Charis , Robinson Bruce G.
Published: November 2003

OBJECTIVE We have determined the frequency of specific mutations in the RET proto‐oncogene in sporadic medullary thyroid carcinomas (MTCs) and correlated the presence or absence of a codon 918 mutation with the clinical characteristics of these tumours.

DESIGN Thirty paraffin‐embedded sporadic MTCs and two frozen MTCs were collected for analysis of specific mutations in the RET proto‐oncogene in codons 609, 611, 618 and 620 (exon 10); 630 and 634 (exon 11); 768 (exon 13); 883 (exon 15) and 918 (exon 16). A novel primer was designed which introduced a restriction site for RsaI in the presence of the specific codon 918 mutation (ATG → ACG) in these tumour samples. A ‘clinical‐genetic’ correlation was performed comparing the presence or absence of the codon 918 mutation with the following clinical characteristics: age at diagnosis, tumour size, presence or absence of metastases, MTC related morbidity, and base line calcitonin levels at diagnosis or most recent follow‐up.

PATIENTS Patients were classified as having sporadic MTC if there was no family history of C‐cell hyperplasia, MTC, phaeochromocytoma or parathyroid disease. Retrospective review of patient records enabled complete clinical data to be obtained in 28 of 32 patients.

MEASUREMENTS Base line calcitonin levels were measured by radioimmunoassay or calcitonin enzyme linked immunoassay. Cysteine codons in exons 10 and 11, specifically codons 609, 611, 618, 620, 630 and 634, were screened for the presence of mutations by sequence analysis. Specific mutations occurring at codons 768, 883 and 918 were screened for by restriction endonuclease digestion of PCR products.

RESULTS The mutation at codon 918ATG → ACG was found in 21 of 32 (66%) MTCs and the mutation at codon 883GCT → TTT was found in one of 32 MTCs. Where possible, the presence of ‘germline‐type’ mutations in codons 609, 611, 618, 620, 630 and 634 were excluded. Ten MTCs did not have a mutation in codons 768, 883 or 918 of the RET proto‐oncogene. The presence or absence of the somatic mutation at codon 918 did not correlate with any of the above clinical characteristics.

CONCLUSION Somatic mutations in the RET proto‐oncogene occur frequently in sporadic MTCs.
ACTH independent Cushing's syndrome occurring in siblings
Authors: Minami Shiro , Sugihara Hitoshi , Sato Jun , Tatsukuchi Atsushi , Sugisaki Yuichi , Sasano Hironobu , Wakabayashi Ichiji
Published: November 2003

Familial Cushing's syndrome due to ACTH independent bilateral macronodular adrenocortical hyperplasia occurring in siblings is reported. The proband was a 69‐year‐old woman who presented with a typical Cushingoid appearance. The serum cortisol level was elevated, with a loss of diurnal rhythm, and the plasma ACTH level was undetectable. Dynamic testing showed no suppression of urinary 17‐OHCS by high dose dexamethasone and no stimulation by metyrapone. An abdominal CT scan showed bilateral adrenal enlargement. The patient died of a subarachnoid haemorrhage, and autopsy revealed a massively thickened adrenal cortex composed of nodules up to 3.5 cm in diameter. A pituitary adenoma was not found. We learned that the patient's elder brother was also diagnosed at 59 years of age with Cushing's syndrome due to bilateral macronodular adrenocortical hyperplasia. His plasma cortisol levels were not suppressed by high dose dexamethasone and the plasma ACTH level was undetectable. Screening of the available family members by administering 1 mg dexamethasone at midnight and performing abdominal CT scan revealed impaired suppressibility of serum cortisol associated with enlarged bilateral adrenal glands in a 64‐year‐old sister and a 54‐year‐old brother. The 64‐year‐old sister was considered as a possible ‘affected’ case in the early stages of development, because the basal level of ACTH was not suppressed and hyperplasia of the bilateral adrenal glands as revealed by CT scan was less evident.
Steroids in human intrauterine fluids of early pregnancy
Authors: Atkinson G. , Campbell D. J. , Cawood M. L. , Oakey R. E.
Published: November 2003

OBJECTIVE Little is known of the hormone environment of the developing early human embryo. We have therefore measured selected steroids in the intrauterine fluids of early pregnancy.

DESIGN Measurement of progesterone, 17α‐hydroxyprogesterone, oestradiol‐17β, testoste rone, androstenedione, cortisol and dehydroepiandrosterone sulphate in matched samples of coelomic fluid, amniotic fluid and maternal serum collected before pregnancy termination from 12 women between 8 and 12 weeks gestation.

RESULTS Mean concentrations of progesterone, oestradiol‐17β and 17α‐hydroxyprogesterone in coelomic fluid were respectively 20, 6 and 2 times greater than in maternal serum and 8, 13 and 2.6 times those in amniotic fluid. Concentrations of testosterone and androstenedione were highest in maternal serum and lowest in amniotic fluid. Cortisol and dehydroepiandrosterone sulphate were found in intrauterine fluids only at the limit of detection but in normal concentrations in maternal serum.

CONCLUSIONS Coelomic fluid contains relatively high concentrations of progesterone, oestradiol‐17β and 17α‐hydroxyprogesterone which may be synthesized locally. Amniotic fluid contains lower concentrations of steroids (other than progesterone) than are found in coelomic fluid or maternal serum. Free diffusion of steroids across the amnion appears limited. This may constitute a mechanism to protect the embryo from unwanted exposure to biologically active steroids.
A comparison between short ACTH and insulin stress tests for assessing hypothalamo‐pituitary‐adrenal function
Authors: Ammari F. , Issa B. G. , Millward E. , Scanlon M. F.
Published: November 2003

OBJECTIVE Insulin‐induced hypoglycaemia is the standard method for assessment of the hypothalamo‐pituitary‐adrenal (HPA) axis of patients with pituitary or hypothalamic disease. It has been claimed that a normal cortisol response to the 30‐minute ACTH stimulation test (AST) obviates the need to perform the insulin stress test (IST) in these patients. The objective of our study was to compare both tests in a group of consecutive patients with pituitary disease.

SUBJECTS AND METHODS Thirty patients with pituitary disease were evaluated by standard IST (0.1 U of soluble insulin/kg body weight,i.v.) after fasting from midnight and AST (250μg synacthen, i.v.). In the IST, a plasma glucose of <2.2mmol/l was taken as the hypoglycaemic threshold and blood was collected at 0, 30, 60, 90 and 120minutes. In the AST blood was collected at 0 and 30minutes. Serum cortisol was measured by standard radioimmunoassay and glucose by the glucose oxidase method. Cortisol responses to the stimuli were compared at cut‐off levels of 550, 500, 450 and 400nmol/l.

RESULTS At 550nmol/l, out of 30 patients, 17 showed an abnormal IST of whom 9 had normal responses to AST (53%). At 500nmol/l, 12 patients had an abnormal IST of whom 6 had normal AST (50%). At 450nmol/l, of 9 patients with an abnormal IST, 5 had a normal AST (56%). At 400nmol/l, 5 patients had an abnormal IST all of whom (100%) showed a normal AST.

CONCLUSION There is a clear discrepancy between the results of the two tests at different cortisol cut‐off levels. The ACTH stimulation test is not reliable for assessing the HPA axis in patients with pituitary disease and the insulin stress test remains the standard method.
Leuprolide suppression of androgen secretion by hilus cell hyperplasia within the wall of an ovarian cyst
Authors: De Santiago Manuel , Herranz Lucrecia , Ordas Juan , Atienza Miguel , Suarez‐Mier Maria , Gancedo Pilar G.
Published: November 2003

Androgenic manifestations coexisting with hilus cell hyperplasia adjacent to a tumour or an ovarian cyst are extremely rare. We report the case of a post‐menopausal woman with hirsutism associated with hilus cell hyperplasia within the wall of an ovarian cyst. The pattern of steroid secretion revealed increased testosterone release. Suppression of testosterone to ‘normal range’ was seen in response to leuprolide administration. This new approach demonstrates gonadotrophin dependence of hilus cell hyperplasia within an ovarian cyst.
Laparoscopic trans‐peritoneal adrenalectomy: a preliminary report of 14 adrenalectomies
Authors: Walmsley David , McIntyre Robert , Sawers Hilary A. , Shaw James A. M. , Webster John , Krukowski Zygmunt H. , Bevan John S.
Published: November 2003

OBJECTIVES Laparoscopic adrenalectomy offers the potential benefits of a smaller operation with more rapid hospital discharge, compared to open surgery. Only a few small series have been reported so far. We describe our preliminary experience of 14 adrenalectomies using this new technique.

DESIGN Review of all adrenalectomies (with the pre‐operative intention of laparoscopic removal) performed in an endocrine unit whose surgeon already had abdominal laparoscopic experience, particularly with cholecystectomy.

PATIENTS AND MEASUREMENTS Twelve patients (3 with Conn's syndrome, 3 Cushing's syndrome, 1 Cushing's disease, 2 phaeochromocytomas and 3 adrenal incidentalomas) were operated between September 1993 and February 1996. Operating times, operative technique, time from surgery to discharge, outcome and all complications were recorded prospectively. Comparative data were obtained from 14 consecutive open adrenalectomies performed by the same surgeon between February 1989 and February 1995.

RESULTS Fourteen glands were removed, two with a cholecystectomy, in 12 operations. Operating time (mean (range) 120 (60–225) min) was reduced with experience. Positioning the patient in the right lateral position facilitated left adrenalectomy. Time to discharge (mean (range)) was 5.3 (1–12) days. There were relatively minor complications in three patients, including two with Cushing's syndrome: a hernia at a port site, intra‐peritoneal/wound haemorrhage and a pressure sore. Time to discharge for open adrenalectomy (mean (range)) was 6.5 (2–11) days and one case was complicated by wound infection.

CONCLUSIONS Laparoscopic adrenalectomy is a practical technique for appropriately trained surgeons who regularly undertake adrenalectomy. The smaller incisions offer potential advantages, particularly for patients with poor tissue quality due to Cushing's syndrome, but tissue haemorrhage may still be a problem in these patients. Time to hospital discharge was similar to that for open surgery.
Loss of heterozygosity studies at the retinoblastoma and breast cancer susceptibility (BRCA2) loci in pituitary, parathyroid, pancreatic and carcinoid tumours
Authors: Pearce S. H. S. , Trump D. , Wooding C. , Sheppard M. N. , Clayton R. N. , Thakker R. V.
Published: November 2003

OBJECTIVE Allelic deletion of the retinoblastoma (Rb) gene on chromosome 13 has been reported in both pituitary and parathyroid tumours. We have investigated the roles of the Rb and the hereditary breast cancer susceptibility gene (BRCA2), which lie within 25 cM of each other on chromosome 13q12–14, in the multi‐step aetiology of endocrine tumours.

PATIENTS AND MEASUREMENTS Seventy‐seven endocrine tumours (43 anterior pituitary, 22 parathyroid, 7 carcinoid, and 5 pancreatic islet cell tumours) with paired leucocytes have been examined for loss of heterozygosity (LOH) at the Rb and BRCA2 loci by using specific oligonucleotide primers for the PCR amplification of microsatellite polymorphisms at three intragenic Rb markers, Rb1.20, Rbi4 and D13S153, and D13S260 which is linked to the BRCA2 locus.

RESULTS Seventy‐five of the 77 tumour–leucocyte pairs were informative and LOH was detected in 1 of 16 non‐functioning pituitary tumours, 1 of 8 prolactinomas, 3 of 19 parathyroid adenomas and 1 of 1 parathyroid carcinoma. All the 3 parathyroid adenomas with LOH were associated with aggressive clinical and histopathological features. Allele loss was not detected in any of the 16 somatotrophinomas, 2 corticotrophinomas, 1 gonadotrophinoma, 7 carcinoid tumours (6 bronchial, 1 metastatic intestinal) or 5 pancreatic islet cell tumours that were informative.

CONCLUSIONS These results demonstrate that allelic deletions of the 13q12‐14 region occur in some pituitary adenomas and 16% of parathyroid adenomas. The extensive loss, which involves both the Rb gene and the BRCA2 locus, suggests that tumour suppressor genes in this region other than Rb or BRCA2 may be involved in the development and progression of some endocrine tumours.
Reversible primary hypothyroidism with blocking or stimulating type TSH binding inhibitor immunoglobulin following recombinant interferon‐α therapy in patients with pre‐existing thyroid disorders
Authors: Chen Fu‐Qin , Okamura Ken , Sato Kaori , Kuroda Takeo , Mizokami Tetsuya , Fujikawa Megumi , Tsuji Hiroshi , Okamura Seiichi , Fujishima Masatoshi
Published: November 2003

OBJECTIVE Treatment with recombinant interferon‐α (rIFN‐α) may induce autoimmunity. We have evaluated the effect of rIFN‐α on pre‐existing thyroid disease with special reference to changes in TSH receptor antibody.

DESIGN AND PATIENTS Five patients, who had a history of autoimmune thyroid disease diagnosed between 2 and 16 years earlier (three patients had Graves' disease while two had Hashimoto's thyroiditis), were treated with rIFN‐α for chronic hepatitis C. Before, during and after rIFN‐α therapy, we determined thyroid function, antithyroid antibody, thyroid echogenicity and the surface phenotype of the peripheral and intrathyroidal lymphocytes.

RESULTS Four of the patients developed overt hypothyroidism after 4–7 months of rIFN‐α therapy, and two of them had a preceding history of low‐uptake thyrotoxicosis. Recovery of thyroid function was observed in all four patients. Strongly positive blocking type TSH receptor antibody was detected and an increase in the percentage of CD19 positive cells in the intrathyroidal lymphocytes was also observed in three of the patients even though the goitre size increased in two of them. One of the patients became thyrotoxic later when stimulating type TSH receptor antibody became positive. Another patient suffered from reversible hypothyroidism although stimulating type TSH receptor antibody remained strongly positive throughout the clinical course.

CONCLUSIONS Our data thus indicated a high incidence of an unusual type of reversible hypothyroidism with TSH receptor antibodies in patients with chronic hepatitis C and pre‐existing autoimmune thyroid disease after recombinant interferon‐α therapy through a mechanism involving both the humoral and cellular immune systems.
Rapid screening for amyloid‐related variant forms of transthyretin is possible by electrospray ionization mass spectrometry
Authors: RANLOslash;V I. , ANDO Y. , OHLSSON P. ‐I. , HOLMGREN G. , RANLOslash;V P. J. , SUHR O. B.
Published: November 2003

We have used a new and rapid method to detect three variant forms of transthyretin (TTR): methionine for valine at position 30 (Met‐30), serine for cysteine at position 6 (Ser‐6) and methionine for leucine at position 111 (Met‐111). By using an anti‐transthyretin antibody and a centrifugal concentrator, transthyretin was isolated from plasma samples and analysed for variant forms by electrospray ionization mass spectrometry. Differentiation between homozygous and heterozygous transthyretin Met‐30 and Met‐111 posed no problems. However, a clear separation of transthyretin Ser‐6 and Met‐111 peaks in one patient with concordant Ser‐6 and Met‐111 mutations could not be achieved. The present method enables a quick and reliable detection of variant TTR. It can be used to screen families or small populations for abnormal TTR. Knowledge of TTR polymorphism and the variable expression of different amyloidogenic mutations should be taken into consideration when applying the methods in clinical practice.
The Asn‐291→Ser and Ser‐477→Stop mutations of the lipoprotein lipase gene and their significance for lipid metabolism in patients with hypertriglyceridaemia
Authors: KNUDSEN P. , MURTOMÄKI S. , ANTIKAINEN M. , EHNHOLM S. , LAHDENPERÄ S. , EHNHOLM C. , TASKINEN M.‐R.
Published: November 2003

We examined 99 Finnish patients whose serum fasting triglycerides (TG) had exceeded 6.0 mmol L−1, with special interest to their lipid, lipoprotein and post‐heparin plasma lipase activities. The control group consisted of 75 healthy individuals. We also determined the frequency of the Asn‐291→Ser and Ser‐447→Stop mutations both in hypertriglyceridaemic (HTG) subjects and in control subjects. A total of 51 of the original 99 hypertriglyceridaemic patients still had TG > 6.0 mmol L−1 when measured a second time. They are referred to as persistently hypertriglyceridaemic subjects (pHTG). The remaining 48 subjects had TG < 6.0 mmol L−1 in the second measurement and are referred to as sporadically hypertriglyceridaemic subjects (sHTG). The allelic frequencies of the Ser‐447→Stop mutation in the total HTG and sHTG groups were similar to the frequencies present in the control group, but lower in pHTG patients compared with the control group (0.049 vs. 0.153, χ2 = 6.63, P < 0.05). The Asn‐291→Ser mutation was more frequent in HTG group than in the control group (0.0606 vs. 0.013, χ2 = 4.86, P < 0.05). This difference was due to the higher frequency of the minor allele of Asn‐291→Ser in the cohort with persistent hypertriglyceridaemia compared with the control group (0.088 vs. 0.013, χ2 = 8.00, P < 0.01 ). The highest frequency (0.114) of the minor allele of Asn‐291→Ser was found in type 2 diabetic patients with persistent hypertriglyceridaemia. The carrier status of Asn‐291→Ser or Ser‐447→Stop did not predict either post‐heparin plasma lipoprotein lipase (LPL) activities or lipid and lipoprotein levels in any of the groups studied. Our data suggest that overproduction of very low‐density lipoproteins (VLDL) is a more important cause of hypertriglyceridaemia in the Finns than is the LPL deficiency.
The effects of salinity on aquatic plant germination and zooplankton hatching from two wetland sediments
Authors: Nielsen Daryl L. , Brock Margaret A. , Crosslé Katharine , Harris Ken , Healey Michael , Jarosinski Irene
Published: November 2003

1. The effect of increasing salinity on the emergence of zooplankton eggs and the germination of aquatic plant seeds from the sediment of two wetlands was examined. Salinity was found to cause reductions in species richness and abundance of aquatic plants and zooplankton at salinities between 1000 and 5000 mg L−1. Aquatic plants also had an associated decrease in above ground biomass.

2. Individual taxa showed different responses to salinity, and four response patterns were identified: (i) increased number of organisms emerging at 1000 mg L−1; (ii) decreased number of organisms emerging above 1000 mg L−1; (iii) decreased number of organisms emerging between 300 and 1000 mg L−1; (iv) no difference in number of organisms emerging across the range of salinities. Response patterns (iii) and (iv) were common to both plants and zooplankton, whereas response patterns (i) and (ii) were only identified for zooplankton.

3. Results indicate that there is potential for the increasing salinity in Australian rivers and wetlands to decrease the species richness of aquatic communities resulting in loss of wetland biodiversity.
Effects of the non‐indigenous cladoceran Cercopagis pengoi on the lower food web of Lake Ontario
Authors: Laxson Corey L. , McPhedran Kerry N. , Makarewicz Joseph C. , Telesh Irena V. , Macisaac Hugh J.
Published: November 2003

1. In North America, the invasive predatory cladoceran Cercopagis pengoi was first detected in Lake Ontario. We explored the impact of Cercopagis on the lower food web of Lake Ontario through assessments of historical and seasonal abundance of the crustacean zooplankton, by conducting feeding experiments on the dominant prey of the invader, and by estimating its food requirements.

2. Between 1999 and 2001, a decrease in the abundance of dominant members of the Lake Ontario zooplankton community (Daphnia retrocurva, Bosmina longirostris and Diacyclops thomasi) coincided with an increase in the abundance of Cercopagis. Daphnia retrocurva populations declined despite high fecundity in all 3 years, indicating that food limitation was not responsible. Chlorophyll a concentration generally increased, concomitant with a decline in the herbivorous cladoceran zooplankton in the lake.

3. Laboratory experiments demonstrated that Cercopagis fed on small‐bodied species including D. retrocurva and B. longirostris.

4. Consumption demand of mid‐summer populations of Cercopagis, estimated from a bioenergetic model of the confamilial Bythotrephes, was sufficient to reduce crustacean abundance, although the degree of expected suppression varied seasonally and interannually.

5. Predatory effects exerted by Cercopagis on the Lake Ontario zooplankton, while initially very pronounced, have decreased steadily as the species became established in the lake.