bluuebunny/crossref_metadata_2025 huggingface dataset:
Partial iodide organification defect caused by a novel mutation of the thyroid peroxidase gene in three siblings
Authors: Kotani Tomio , Umeki Kazumi , Kawano Jun–ichi , Suganuma Tatsuo , Hishinuma Akira , Ieiri Tamio , Harada Shohei
Published: July 2003

background Three siblings with goitre and latent to mild hypothyroidism were suspected of having thyroid peroxidase (TPO) abnormality. Direct sequencing of their genomic DNAs showed two novel mutations of the TPO gene, one of which was G1687T (Gly533Cys; exon 9) and the other 1808–13del (Asp574/Leu575del; exon 10). The two mutations were compound heterozygous, as the former was found in their father's DNA as heterozygous, and the latter was found in DNA from their mother, also as heterozygous. As Gly533 and Asp574/Leu575 were well‐conserved amino acids in the peroxidase superfamily, Gly533Cys‐ and Asp574/Leu575del‐TPOs were thought to be affected structurally or functionally. In expression studies using CHO‐Kl cells and mRNAs introduced with individual mutations, both mutated TPO proteins were expressed at the same molecular size as wild‐type TPO and had enzyme activity, although Gly533Cys‐TPO was slightly lower in efficiency of expression and more degenerative than wild‐type TPO.

methods We examined the localization of both mutated TPOs. Gly533Cys‐TPO was located on the endoplasmic reticulum (ER) and nuclear envelope but not on the plasma membrane, whereas Asp574/Leu575del‐TPO was located not only on the ER and nuclear envelope but also on the plasma membrane, as wild‐type TPO. Nevertheless, only one point differed between Asp574/Leu575del‐ and wild‐type TPOs: the mutated TPO was expressed on the plasma membrane surface at less than half the rate of wild‐type TPO.

results Gly533Cys‐TPO synthesized almost no thyroid hormone because of its defective localization on the apical membrane surface of thyrocytes, whereas Asp574/Leu575del‐TPO performed thyroid hormone synthesis at a rate of less half that of wild‐type TPO. In cotransfection experiments using three combinations of wild‐type and G1687T‐mRNAs, wild‐type and 1808–13del‐mRNAs, and G1687T‐, 1808–13del‐mRNAs, the three kinds of mRNAs were considered to have no influence on cell surface TPO expression of another mRNA when a 50%‐maximal amount of each mRNA was transfected. When a larger amount of each mRNA was transfected, the former two combinations showed the level of cell surface TPO expression obtained from the saturating amount of wild‐type mRNA, whereas the last combination of mutated mRNAs covered only about half of the expression level.

conclusion Defective thyroid hormone production resulting from the abnormal TPOs was at a level that caused latent hypothyroidism when the patients were born. With their growth, thyroid hormone volume gradually became inadequate and their thyroid gland enlarged compensatorily.
Grey‐scale analysis allows a quantitative evaluation of thyroid echogenicity in the patients with Hashimoto's thyroiditis
Authors: Mazziotti Gherardo , Sorvillo Francesca , Iorio Sergio , Carbone Antonella , Romeo Antonio , Piscopo Marco , Capuano Salvatore , Capuano Ermanno , Amato Giovanni , Carella Carlo
Published: July 2003

objective In the present study we have performed a grey‐scale quantitative analysis of thyroid echogenicity in the patients affected by Hashimoto's thyroiditis (HT), obtaining a numerical estimate of the degree of hypoecogenicity associated with the appearance of thyroid dysfunction.

materials and methods The study group included 89 patients with serum positivity for thyroglobulin (TgAb) and/or peroxidase (TPOAb) antibodies. Ultrasound (US) evaluation of thyroid gland and biochemical assay of serum thyrotropin (TSH), free‐thyroxine (FT4) and free‐triiodiothyronyne (FT3) were performed in all patients, and in 40 healthy subjects enrolled as control group. Thyroid echogenicity was compared with that of the surrounding neck muscles, using the grey‐scale histogram analysis. The echogenicity was expressed in grey‐scales (GWE).

results In HT patients, the mean of thyroid echogenicity was lower when compared to the normal thyroid (61·9 ± 8·3 GWE vs. 71·9 ± 3·1 GWE; P = 0·01). In all HT patients the lowest limit of thyroid echo distribution was in the echogenicity range of the surrounding muscle, the overlapping ranging between 3·4% and 95·0% (mean ± SD 48·4 ± 20·9%). The extension of like‐muscle hypoechogenicity into the thyroid gland was significantly correlated with serum TSH values (r = 0·37; P < 0·001), serum FT4 values (r = –0·60; P < 0·001), and serum TPOAb values (r = 0·31; P = 0·004). Nobody was hypothyroid when the hypoechogenicity was less than 38·0%, whereas hypothyrodism occurred in all cases with hypoechogenicity of more than 68·9%. The receiving operating characteristic curve demonstrated that 48·3% was the best cut‐off for identifying hypothyroid patients with sensitivity, specificity and diagnostic accuracy of 88·9%, 86·3% and 87·6%, respectively.

conclusions In conclusion, the grey‐scale quantitative analysis has provided a measure of thyroid hypoechogenicity associated with the appearance of hypothyroidism during the course of HT. The results of the present study would encourage the application of the computerized grey‐scale analysis as complementary tool to US evaluation in the patients affected by HT.
The N363S polymorphism in the glucocorticoid receptor gene is associated with overweight in subjects with type 2 diabetes mellitus
Authors: Roussel Ronan , Reis André F. , Dubois‐Laforgue Danièle , Bellanné‐Chantelot Christine , Timsit José , Velho Gilberto
Published: July 2003

objective A single nucleotide polymorphism (A1220G; N363S) in exon 2 of the glucocorticoid receptor gene (NR3C1), associated with increased sensitivity to glucocorticoids, was shown to be associated with obesity in nondiabetic subjects. Here, we investigated the impact of this variant on traits related to obesity and hyperglycaemia in subjects with type 2 diabetes mellitus.

patients and measurements The N363S variant was screened by restriction fragment length polymorphism technique following DNA amplification by polymerase chain reaction in 369 French Caucasians with type 2 diabetes mellitus.

results Twenty subjects were found to be heterozygous for the variant (AG genotype frequency 0·0542). The prevalence of overweight [body mass index (BMI) > 25 kg/m2] was higher in AG carriers than in AA carriers (100%vs. 73%, P = 0·003). Moreover, the mean body weight and the BMI were higher in AG as compared to AA carriers, although only the body weight was significantly different between groups. However, when only the men were considered, a significantly higher BMI was observed in AG as compared to AA carriers: 30·0 ± 4·8 vs. 27·3 ± 4·6 kg/m2 (BMI Z‐score 1·28 ± 1·38 vs. 0·55 ± 1·17; P = 0·035). No evidence was found for an association of the N363S variant with parameters related to the severity of hyperglycaemia.

conclusions The 363S allele of the N363S variant of NR3C1 is associated with the susceptibility to overweight in subjects with type 2 diabetes mellitus.
Apolipoprotein E3Basel: new insights into a highly conserved protein region
Authors: Miserez A. R. , Scharnagl H. , Muller P. Y. , Mirsaidi R. , Stähelin H. B. , Monsch A. , März W. , Hoffmann M. M.
Published: July 2003

Background Apolipoprotein E is important for the receptor‐mediated uptake of triglyceride‐rich lipoproteins. Mutations in the gene encoding apolipoprotein E may cause a reduced uptake of these lipoproteins. Particular apolipoprotein E mutations have been also found to be associated with nephrologic, neurologic, and even ophthalmologic diseases. Hence, a continuously expanding role in biology is being attributed to this protein.

Design Randomly selected volunteers from of a large Swiss cohort were genotyped for the common apolipoprotein E isoforms (apolipoprotein E2, apolipoprotein E3, apolipoprotein E4).

Results In one of the volunteers, a novel C‐to‐T mutation causing an alanine‐to‐valine substitution (A106V, designated apolipoprotein E3Basel) was discovered. Alanine at residue 106 is highly conserved between mammalian species and is located in the immediate vicinity of the 112C/R polymorphism (apolipoprotein E4). Recombinant apolipoprotein E3Basel, expressed in the baculovirus system, displayed no detectable reduction in its low density lipoprotein (LDL) receptor‐ and heparin‐binding activities. Despite normal binding functions, apolipoprotein E3Basel might cause modifications in the lipoprotein pattern. In the index case, plasma triglycerides were elevated and in two further apolipoprotein E3Basel‐carriers, cholesterol, phospholipid, apolipoprotein CIII levels, LDL‐cholesterol/apoB‐100‐ and VLDL‐triglyceride/VLDL‐cholesterol‐ratios were higher compared with apolipoprotein E3Basel‐noncarriers when pair‐matched for age and gender. One of the four apolipoprotein E3Basel‐carriers from the index family had a personal history of Alzheimer's disease.

Conclusions Alanine at amino acid position 106 is highly conserved but not crucial in the receptor‐mediated uptake of lipoprotein particles. Nevertheless, amino acid position 106 might be involved in the apolipoprotein E‐dependent regulation of the lipoprotein lipase that hydrolyzes triglycerides and in the development of Alzheimer's disease.
Acute‐phase response of human hepatocytes after infection with Chlamydia pneumoniae and cytomegalovirus
Authors: Verkerk M. S. A. , Visseren F. L. J. , Paul Bouter K. , Diepersloot R. J. A.
Published: July 2003

Background There is increasing evidence that chronic inflammation plays a pivotal role in the development of atherosclerosis. Whether inflammation is the cause or consequence of vascular damage is unclear. Also, the source of inflammation is unknown, but may well be infection by Cytomegalovirus (CMV) or Chlamydia pneumoniae (C. pneumoniae). Infection of the liver by CMV or C. pneumoniae may induce a general inflammatory reaction contributing to accelerated atherogenesis.

In this study we investigated the production of interleukin‐6 (IL‐6), fibrinogen and plasminogen activator inhibitor‐1 (PAI‐1) by hepatocytes after infection with CMV or C. pneumoniae.

Methods HepG2 cell monolayers were grown to confluence in 48‐well tissue culture plates. Hepatocytes were infected with 50 µL or 100 µL of suspension of CMV (102·70 TCID50 mL−1) and C. pneumoniae (104·75 TCID50 mL−1). The medium of the inoculated cells was collected every 24 h, from day 1 to day 4, for determination of IL‐6, PAI‐1 and fibrinogen concentrations.

Results Fibrinogen production was increased significantly in a dose‐dependent manner after infection with CMV (50 µL: P = 0·022 and 100 µL: P < 0·001) and C. pneumoniae (P = 0·012). Cytomegalovirus infection resulted in an increase of IL‐6 production compared with uninfected cells (P = 0·048). Cytomegalovirus and C. pneumoniae infection did not result in a significantly increase of PAI‐1 production by hepatocytes.

Conclusion We conclude that in addition to direct vascular wall infection by C. pneumoniae and CMV, virus‐related development of atherosclerosis might also be initiated by chronic liver infection and subsequent production of inflammatory and procoagulant mediators released in the circulation. This may be another pathophysiological link for the observed relation between infections and the development of atherosclerosis.
Relation between insulin kinetics and insulin sensitivity in pregnancy
Authors: Jolly M. C. , Hovorka R. , Godsland I. , Amin R. , Lawrence N. , Anyaoku V. , Johnston D. , Robinson S.
Published: July 2003

Background Different time‐concentration profiles of plasma insulin following insulin modification of a frequently sampled intravenous glucose‐tolerance‐test (FSIVGTT) were observed in a study investigating maternal metabolism and fetal macrosomia. We aimed to investigate whether these differences were related to the volume of distribution of insulin, insulin clearance, or both.

Design Forty‐four women were studied between 33 and 35 weeks’ gestation using an insulin‐modified FSIVGTT. Specific insulin was assayed with an enzyme‐linked immunosorbent assay. Insulin sensitivity was calculated using the minimal model and the homeostasis model assessment (HOMA). The volume of distribution and clearance of insulin were calculated from measurements between 2 and 155 min after insulin modification using a one‐compartment model.

Results In accordance with the method for deriving the volume of distribution, there was a significant negative correlation between the increment in insulin concentration and the volume of distribution (rho = −0·92, P < 0·0001). The insulin increment was also related negatively to the clearance of insulin (rho = −0·88, P < 0·0001). There was a significant correlation between the volume of distribution and both the insulin sensitivity index (rho = 0·56, P < 0·0001) and HOMA‐%S (rho = 0·30, P= 0·048), and between the clearance of insulin and both the insulin sensitivity index (rho = 0·83, P < 0·0001)) and HOMA‐%S (rho = 0·34, P= 0·025).

Conclusion The different time‐concentration profiles of plasma insulin resulted from differences in the volume of distribution and clearance of insulin. There was a correlation between insulin kinetics and the insulin sensitivity index. Further research is required to investigate possible mechanisms by which insulin kinetics may be related to insulin sensitivity.
Predictors of lipoprotein(a) levels in a European and South Asian population in the Newcastle Heart Project
Authors: Tavridou A. , Unwin N. , Bhopal R. , Laker M. F.
Published: July 2003

Background Understanding of the higher susceptibility of South Asians to coronary heart disease is limited. One explanation is the combination of high prevalence of insulin resistance with higher lipoprotein(a) levels.

Materials and methods Lipoprotein(a) levels and genotypes in three South Asian groups aged 25–74 years (Indian, Pakistani, Bangladeshi) were compared with a European population in a cross‐sectional study. Biochemical measurements included lipids, apolipoprotein A1 and B, glucose, insulin and fibrinogen. Insulin sensitivity was calculated using the homoeostasis model assessment method (HOMA).

Results There was no significant difference in lipoprotein(a) levels between South Asian and European men. South Asian women combined had higher lipoprotein(a) levels than European women, a difference probably resulting from higher lipoprotein(a) levels in Pakistani women compared with Indian and Bangladeshi women. Fasting insulin and HOMA were negatively associated with Lp(a) in South Asians though the associations were statistically significant only in men. There were only modest associations between most cardiovascular risk factors and Lp(a). Twenty‐seven apolipoprotein(a) size alleles were detected in the three South Asian groups ranging from 16 to 43 kringle‐IV repeats. The apolipoprotein(a) size polymorphism explained 23% of the variability in lipoprotein(a) levels in South Asians.

Conclusions There were few nongenetic predictors of lipoprotein(a) levels in South Asians and Europeans. The lack of difference in Lp(a) between the South Asian and European men and the fact that differences between the women seemed to be confined to the Pakistani group offer little support to the hypothesis that higher Lp(a) levels contribute to the increased risk of heart disease in South Asians. Our findings do not support the hypothesis that susceptibility to heart disease in South Asians results from a combination of high insulin resistance and high Lp(a) levels.
The evolution of HOM‐C homeoboxes in the Dipteran family Drosophilidae
Authors: DeSalle R. , Branham M. A. , O'Grady P. , Gatesy J.
Published: July 2003

Forty‐five new Homeotic Complex (HOM‐C) homeobox sequences from six species of Drosophilidae (Drosophila heteroneura, D. adiastola, Zaprionus vittiger, Chymomyza amoena, Scaptodrosophila pattersoni and Hirtodrosophila pictiventris) were obtained using a PCR‐cloning method. These new homeoboxes are from the labial, proboscipedia, Deformed, Sex combs reduced, fushi tarazu, Antennapedia, Ultrabithorax, abdominal‐A and Abdominal‐B genes. Phylogenetic signal in the homeobox sequences was assessed and several aspects of sequence evolution were examined. In particular, codon bias was examined and found to exist between the drosophilid species examined here and Anopheles gambiae outgroup sequences. In addition, different patterns of codon bias were detected in homeoboxes interrupted with introns when compared to homeoboxes that are uninterrupted.
Stress response in a juvenile hormone‐deficient Drosophila melanogaster mutant apterous56f
Authors: Gruntenko N. E. , Chentsova N. A. , Andreenkova E. V. , Bownes M. , Segal D. , Adonyeva N. V. , Rauschenbach I. Yu
Published: July 2003

The apterous56f (ap56f) mutation leads to increases in juvenile hormone (JH) degradation levels and JH‐esterase makes a greater contribution to the increase than JH‐epoxide hydrolase. Dopamine levels in ap56f females, but not males, are higher than in wild‐type. JH treatment of ap56f and wild‐type females decreases their dopamine levels. ap56f females, but not males, produce less progeny. Survival under heat stress is dramatically decreased in ap56f females, but not males. ap56f flies show a stress reaction, as judged by changes in tyrosine decarboxylase and JH‐hydrolysing activities, dopamine levels and fertility, but its intensity in the mutant females, but not males, differs significantly from wild‐type. Thus, the ap56f mutation causes dramatic changes in female, but not male, metabolism and fitness.
Characterization of an intestinal mucin from the peritrophic matrix of the diamondback moth, Plutella xylostella
Authors: Sarauer B. L. , Gillott C. , Hegedus Dwayne
Published: July 2003

The peritrophic matrix (PM) of Plutella xylostella larvae was found to contain twelve integral and eighteen loosely associated proteins. An antiserum against Mamestra configurata integral PM proteins cross‐reacted with several P. xylostella PM proteins and was used to isolate a partial cDNA encoding an insect intestinal mucin (PxIIM). PxIIM was expressed primarily in the larval midgut. The deduced protein sequence of the partial cDNA contained three potentially glycosylated, mucin‐like domains and six cysteine‐rich chitin‐binding domains (CBDs). An additional chitin‐binding domain was proposed to reside at the amino terminus of the protein based on comparison with other IIM. The organization of mucin domains and CBDs exhibited features, including an internal triplet of regularly spaced CBDs and a carboxyl terminal CBD with two additional conserved cysteine residues, that were found to be common to other lepidopteran IIMs.
Fat body expressed yolk protein genes in Hyphantria cunea are related to the YP4 follicular epithelium yolk protein subunit gene of pyralid moths
Authors: Cheon H. M. , Kim H. J. , Yun C. Y. , Lee H. J. , Lee I. H. , Shirk P. D. , Seo S. J.
Published: July 2003

cDNA clones for two of the yolk proteins, YP1 and YP2, produced by the fat body of the moth, Hyphantria cunea, were sequenced and found to be homologous to the follicular epithelium yolk proteins of pyralid moths. Both cDNA clones coded for polypeptides of 290 residues and the deduced amino acid sequence identity between YP1 and YP2 was very high (79.0%). Analysis of the secondary structure of the predicted polypeptides suggests that YP1 and YP2 do not form heteromeric proteins because of differences in secondary structure due to the lack of alpha helices in YP1. Northern blot analysis showed that the transcripts for YP1 (1.2 kb) and YP2 (1.1 kb) were present primarily in the female fat body with only trace levels detectable in the ovary of the adult female. In a developmental study, the YP1 and YP2 transcripts were first detectable in 10‐day‐old pupae and increased into the adult stage. These results suggest that the YP1 and YP2 genes in H. cunea have been recruited to replace the vitellogenin gene as the primary source of yolk proteins. During this process they have acquired a modified pattern of expression that is different from homologous genes reported in pyralid moths. The assessment of the evolution of proteinaceous yolk in these moths should serve as an excellent model for the evolution of gene recruitment.
Heat stress affects oogenesis differently in wild‐type Drosophila virilis and a mutant with altered juvenile hormone and 20‐hydroxyecdysone levels
Authors: Gruntenko N. E. , Bownes M. , Terashima J. , Sukhanova M. Zh. , Raushenbach I. Yu
Published: July 2003

The link between reproduction and environmental signals is poorly understood at the physiological, genetic and molecular levels. We describe a mutant strain of Drosophila virilis that has altered responses to heat stress. Heat stress in wild‐type females results in oocyte maturation delays, degradation of early vitellogenic egg chambers, inhibition of yolk protein gene expression in follicle cells and accumulation of mature oocytes. The mutant females have increased levels of ecdysteroids and decreased juvenile hormone degradation, and show all of the heat‐stress‐induced reproductive effects observed in wild‐type flies, without exposure to heat stress. During oogenesis in mutant females following heat stress there is an increase in early vitellogenic oocyte degradation and some degradation of late egg chambers. 20‐Hydroxyecdysone levels, but not juvenile hormone degradation, change following heat stress in mutant females.
Swarming motility in undomesticated Bacillus subtilis
Authors: Kearns Daniel B. , Losick Richard
Published: July 2003

Swarming motility was identified and characterized in an undomesticated strain of Bacillus subtilis. Rapid surface migration was preceded by a cell density‐dependent lag period, which could be eliminated if actively swarming cells were used as the inoculum. The leading edge of the swarm was characterized by multicellular rafts of highly flagellated cells. Flagellum biosynthesis and surfactant production were required for swarming. Swarming was not found in any of several standard laboratory strains. Laboratory strains are characteristically unable to produce surfactant, but such a strain remained unable to swarm even when surfactant was provided by extracellular complementation. We conclude that robust swarming is a feature of undomesticated B. subtilis and that this behaviour has been lost or attenuated in laboratory strains through the accumulation of multiple genetic defects.
MxiK and MxiN interact with the Spa47 ATPase and are required for transit of the needle components MxiH and MxiI, but not of Ipa proteins, through the type III secretion apparatus of Shigella flexneri
Authors: Jouihri Noureddine , Sory Marie‐Paule , Page Anne‐Laure , Gounon Pierre , Parsot Claude , Allaoui Abdelmounaaïm
Published: July 2003

The type III secretion (TTS) pathway is used by numerous Gram‐negative pathogens to inject virulence factors into eukaryotic cells. The Shigella flexneri TTS apparatus (TTSA) spans the bacterial enveloppe and its assembly requires the products of approximately 20 mxi and spa genes. We present a functional analysis of the mxiK, mxiN and mxiL genes. Inactivation of mxiK and mxiN, but not mxiL, resulted in the assembly of a non‐functional TTSA that lacked the outer needle. The amounts of needle components MxiH and MxiI were drastically reduced in mxiK and mxiN mutants and in the secretion defective spa47 mutant, indicating that MxiH and MxiI are degraded if they do not transit through the TTSA. Remarkably, expression of MxiH‐His in the mxiN mutant and MxiI‐His in the mxiK mutant restored assembly of a functional TTSA, as shown by the ability of these strains to enter into epithelial cells and to secrete Ipa proteins in response to activation by Congo red. Using a two‐hybrid screen in yeast and immunoprecipitation assays from S. flexneri extracts, we identified interactions between MxiK and Spa33 and Spa47 and between MxiN and Spa33 and Spa47. These results suggest that transit of the needle components MxiH and MxiI through the TTSA involves the concerted action of the cytoplasmic proteins Spa47, Spa33, MxiK and MxiN. They also show that neither MxiK nor MxiN are absolutely required for secretion of Ipa proteins, provided that the TTSA is correctly assembled.
LexA‐independent DNA damage‐mediated induction of gene expression in Myxococcus xanthus
Authors: Campoy Susana , Fontes Marta , Padmanabhan S. , Cortés Pilar , Llagostera Montserrat , Barbé Jordi
Published: July 2003

Myxococcus xanthus, a member of the Proteobacteria delta‐class, has two independent recA genes, recA1 and recA2, but only recA2 is DNA damage‐inducible. The lexA gene has been isolated from M. xanthus by PCR amplification with oligonucleotides designed after sequence identification by tblastn analysis of its genome at the Cereon Microbial Sequence Database. The M. xanthus purified LexA protein is shown to bind specifically to the consensus sequence CTRHAMRYBYGTTCAGS present upstream of lexA and recA2. A degenerate copy of this motif but with important differences can be identified in the region upstream of the recA1 gene. A knock‐out lexA(Def) mutant that has been generated does not differ significantly from wild type in morphology, growth rate, light‐induced carotenogenesis or development. Using transcriptional lacZ fusions and quantitative RT‐PCR analysis, it has been demonstrated that expression of both lexA and recA2 genes is constitutive in the lexA(Def) mutant, whereas the transcription of the DNA damage non‐inducible recA1 gene is not affected in this strain. recN and ssb, whose expression in Escherichia coli are LexA‐regulated, are induced by DNA damage in the M. xanthus lexA(Def) mutant. These data reveal the existence of different regulatory mechanisms for DNA damage‐inducible genes in bacteria belonging to different phyla.
Regulation of the expression of gas vesicle genes in Haloferax mediterranei: interaction of the two regulatory proteins GvpD and GvpE
Authors: Zimmermann Peter , Pfeifer Felicitas
Published: July 2003

The gas vesicle formation in Haloferax mediterranei occurs in the stationary growth phase and involves the 14 genes mc‐gvpACNO and mc‐gvpDEFGHIJKLM. The appearance of the two regulatory proteins GvpD and GvpE, and also of GvpF, was investigated during the growth of H. mediterranei. GvpD was only found during the stationary growth phase, GvpE was present from the late exponential to stationary growth phase, and GvpF was present only during the exponential growth, although the three genes were co‐transcribed. The impact of GvpD and GvpE on the activity of the promoter of the mc‐gvpACNO gene cluster encoding the gas vesicle structural proteins was analysed in H. volcanii transformants containing the mc‐gvpA gene or a fusion of the mcA promoter with the bgaH reading frame encoding a halobacterial β‐galactosidase as reporter. The experiments proved that GvpE is a transcriptional activator, whereas GvpD is involved in the repression. Protein–protein affinity chromatography was used to search for putative binding partners of GvpD and GvpE. Both proteins were synthesized in Escherichia coli as his‐tagged proteins, isolated under denaturing conditions and refolded by dialysis against buffers containing decreasing urea and increasing KCl concentrations up to 2.5 M. The Ni‐NTA matrix tagged with GvpD‐his or GvpE‐his was incubated with soluble proteins of gas vesicle producing H. mediterranei cells. A 21 kDa protein was purified using the matrix tagged with GvpD‐his which proved to be GvpE by Western analysis. Vice versa, GvpD was purified using the GvpE‐his‐Ni‐NTA matrix. These results strongly suggested that GvpD and GvpE were able to interact and might constitute a regulatory system.
Salmonella type III effectors PipB and PipB2 are targeted to detergent‐resistant microdomains on internal host cell membranes
Authors: Knodler Leigh A. , Vallance Bruce A. , Hensel Michael , Jäckel Daniela , Finlay B. Brett , Steele‐Mortimer Olivia
Published: July 2003

The intracellular pathogen, Salmonella enterica, translocates type III effectors across its vacuolar membrane into host cells. Herein we describe a new Salmonella effector, PipB2, which has sequence similarity to another type III effector, PipB. In phagocytic cells, PipB2 localizes to the Salmonella‐containing vacuole (SCV) and tubular extensions from the SCV, Salmonella‐induced filaments (Sifs). We used the specific targeting of PipB2 in macrophages to characterize Sifs in phagocytic cells for the first time. In epithelial cells, PipB2 has a unique localization pattern, localizing to SCVs and Sifs and additionally to vesicles at the periphery of infected cells. We further show that the N‐terminal 225‐amino‐acid residues of PipB2 are sufficient for type III translocation and association with SCVs and Sifs, but not peripheral vesicles. Subcellular fractionation demonstrated that both PipB and PipB2 associate with host cell membranes and resist extraction by high salt, high pH and to a significant extent, non‐ionic detergent. Furthermore, PipB and PipB2 are enriched in detergent‐resistant microdomains (DRMs), also known as lipid rafts, present on membranes of SCVs and Sifs. The enrichment of Salmonella effectors in DRMs on these intracellular membranes probably permits specific interactions with host cell molecules that are concentrated in these signalling platforms.
The fimbrial adhesin F17‐G of enterotoxigenic Escherichia coli has an immunoglobulin‐like lectin domain that binds N‐acetylglucosamine
Authors: Buts Lieven , Bouckaert Julie , De Genst Erwin , Loris Remy , Oscarson Stefan , Lahmann Martina , Messens Joris , Brosens Elke , Wyns Lode , De Greve Henri
Published: July 2003

The F17‐G adhesin at the tip of flexible F17 fimbriae of enterotoxigenic Escherichia coli mediates binding to N‐acetyl‐β‐d‐glucosamine‐presenting receptors on the microvilli of the intestinal epithelium of ruminants. We report the 1.7 Å resolution crystal structure of the lectin domain of F17‐G, both free and in complex with N‐acetylglucosamine. The monosaccharide is bound on the side of the ellipsoid‐shaped protein in a conserved site around which all natural variations of F17‐G are clustered. A model is proposed for the interaction between F17‐fimbriated E. coli and microvilli with enhanced affinity compared with the binding constant we determined for F17‐G binding to N‐acetylglucosamine (0.85 mM−1). Unexpectedly, the F17‐G structure reveals that the lectin domains of the F17‐G, PapGII and FimH fimbrial adhesins all share the immunoglobulin‐like fold of the structural components (pilins) of their fimbriae, despite lack of any sequence identity. Fold comparisons with pilin and chaperone structures of the chaperone/usher pathway highlight the central role of the C‐terminal β‐strand G of the immunoglobulin‐like fold and provides new insights into pilus assembly, function and adhesion.
RcsCDB His‐Asp phosphorelay system negatively regulates the flhDC operon in Escherichia coli
Authors: Francez‐Charlot Anne , Laugel Bruno , Van Gemert Alice , Dubarry Nelly , Wiorowski Florent , Castanié‐Cornet Marie‐Pierre , Gutierrez Claude , Cam Kaymeuang
Published: July 2003

The genes involved in flagellum synthesis, motility and chemotaxis in Escherichia coli are expressed in a hierarchical fashion. At the top of the hierarchy lies the master regulator FlhDC, required for the expression of the whole set of genes. The operon flhDC is controlled by numerous regulators including H‐NS, CRP, EnvZ/OmpR, QseBC and LrhA. In the present work, we report that the flhDC operon is also negatively regulated by the His‐Asp phosphorelay system RcsCDB. The regulation is potentiated by the RcsB cofactor RcsA. Genetic analysis indicates that an RcsAB box, located downstream of the promoter, is required for the regulation. The binding of RcsB and RcsA to this site was demonstrated by gel retardation and DNase I protection assays. In addition, mutation analysis suggests that RcsA‐specific determinants lie in the right part of the ‘RcsAB box’.
DnaA Protein of Escherichia coli: oligomerization at the E. coli chromosomal origin is required for initiation and involves specific N‐terminal amino acids
Authors: Simmons Lyle A. , Felczak Magdalena , Kaguni Jon M.
Published: July 2003

Iterated DnaA box sequences within the replication origins of bacteria and prokaryotic plasmids are recognized by the replication initiator, DnaA protein. At the E. coli chromosomal origin, oriC, DnaA is speculated to oligomerize to initiate DNA replication. We developed an assay of oligomer formation at oriC that relies on complementation between two dnaA alleles that are inactive by themselves. One allele is dnaA46; its inactivity at the non‐permissive temperature is due to a specific defect in ATP binding. The second allele, T435K, does not support DNA replication because of its inability to bind to DnaA box sequences within oriC. We show that the T435K allele can complement the dnaA46(Ts) allele. The results support a model of oligomer formation in which DnaA box sequences of oriC are bound by DnaA46 to which T435K then binds to form an active complex. Relying on this assay, leucine 5, tryptophan 6 and cysteine 9 in a predicted alpha helix were identified that, when altered, interfere with oligomer formation. Glutamine 8 is additionally needed for oligomer formation on an oriC‐containing plasmid, suggesting that the structure of the DnaA‐oriC complex at the chromosomal oriC locus is similar but not identical to that assembled on a plasmid. Other evidence suggests that proline 28 of DnaA is involved in the recruitment of DnaB to oriC. These results provide direct evidence that DnaA oligomerization at oriC is required for initiation to occur.
Spatial and temporal organization of replicating Escherichia coli chromosomes
Authors: Lau Ivy F. , Filipe Sergio R. , Søballe Britta , Økstad Ole‐Andreas , Barre Francois‐Xavier , Sherratt David J.
Published: July 2003

The positions of DNA regions close to the chromosome replication origin and terminus in growing cells of Escherichia coli have been visualized simultaneously, using new widely applicable reagents. Furthermore, the positions of these regions with respect to a replication factory‐associated protein have been analysed. Time‐lapse analysis has allowed the fate of origins, termini and the FtsZ ring to be followed in a lineage‐specific manner during the formation of microcolonies. These experiments reveal new aspects of the E. coli cell cycle and demonstrate that the replication terminus region is frequently located asymmetrically, on the new pole side of mid‐cell. This asymmetry could provide a mechanism by which the chromosome segregation protein FtsK, located at the division septum, can act directionally to ensure that the septal region is free of DNA before the completion of cell division.
Consumer‐resource stoichiometry in detritus‐based streams
Authors: Cross Wyatt F. , Benstead Jonathan P. , Rosemond Amy D. , Bruce Wallace J.
Published: July 2003

Stoichiometric relationships between consumers and resources in detritus‐based ecosystems have received little attention, despite the importance of detritus in most food webs. We analysed carbon (C), nitrogen (N), and phosphorus (P) content of invertebrate consumers, and basal food resources in two forested headwater streams (one reference and the other nutrient‐enriched). We found large elemental imbalances between consumers and food resources compared with living plant‐based systems, particularly in regard to P content, which were reduced with enrichment. Enrichment significantly increased nutrient content of food resources (consistent with uptake of N and P by detritus‐associated microbes). P content of some invertebrates also increased in the enriched vs. reference stream, suggesting deviation from strict homeostasis. Nutrient content varied significantly among invertebrate functional feeding groups, orders and, to some extent, size classes. Future application of stoichiometric theory to detritus‐based systems should consider the potential for relatively large consumer‐resource elemental imbalances and P storage by insect consumers.
Reconsidering diversity–productivity relationships: directness of productivity estimates matters
Authors: Groner Elli , Novoplansky Ariel
Published: July 2003

Despite extensive research efforts, the controversy over diversity–productivity (D–P) patterns in natural communities still looms large. Recent meta‐analyses suggest that unimodal D–P relationships tend to pre‐dominate in plant studies, while positively linear relationships are more common in animal studies. These patterns, however, are based on studies in which productivity is estimated either directly, based on the biomass or energy of the studied organisms, or indirectly, according to the productivity of lower trophic levels, and various surrogates. Our analysis shows that the distribution of D–P patterns is sensitive to the directness of productivity estimates in animal studies but not in plant studies. Analysis of D–P patterns should be based on direct productivity estimates of the studied organisms, especially in comparative meta‐analyses of communities from multiple trophic levels, where productivity is often affected nonlinearly by indirect factors or when complex feedback interactions are expected between productivity and diversity.
Scale‐dependent interactions between intrinsic and extrinsic processes reduce variability in protist populations
Authors: Orland Mary C.
Published: July 2003

Interactions between intrinsic processes and extrinsic fluctuations can positively impact population persistence in ways often not predicted by classic ecological models. These interactions only arise when the intrinsic and extrinsic processes operate on the proper relative scales in time or space. Both metapopulation theory and resonance/attenuation theory suggest that interactions which lower population variability will occur when the intrinsic and extrinsic process occur on similar time scales. I performed an aquatic protist microcosm experiment to investigate how the relative frequencies of extrinsic density perturbations and intrinsic resource pulses impacted population variability. Population variability was lowest in the treatments of intermediate frequency, in which the extrinsic fluctuations and intrinsic processes were on the same time scale. This result is consistent with general theoretical predictions, and empirically documents the importance of considering scale in interactions between intrinsic and extrinsic processes that positively impact population persistence.
Neighbour‐regulated mortality: the influence of positive and negative density dependence on tree populations in species‐rich tropical forests
Authors: Peters Halton A.
Published: July 2003

Density‐dependent mortality has long been posited as a possible mechanism for the regulation of tropical forest tree density. Despite numerous experimental and phenomenological investigations, the extent to which such mechanisms operate in tropical forests remains unresolved because the demographical signature of density dependence has rarely been found in extensive investigations of established trees. This study used an individual‐based demographical approach to investigate the role of conspecific and heterospecific neighbourhood crowding on tree mortality in a Panamanian and a Malayan tropical forest. More than 80% of the species investigated at each site were found to exhibit density‐dependent mortality. Furthermore, most of these species showed patterns of mortality consistent with the Janzen–Connell hypothesis and the rarely explored hypothesis of species herd protection. This study presents some of the first evidence of species herd protection operating in tree communities.
Biological invasions drive size increases in marine and estuarine invertebrates
Authors: Grosholz Edwin D. , Ruiz Gregory M.
Published: July 2003

Ecologists have long been fascinated by the morphological changes that species frequently undergo when introduced into new regions. In this study an unusual pattern of size change associated with the invasion of 19 species of marine and estuarine invertebrates is reported. The results show that the majority of species are significantly larger in the introduced range compared with the native range with little evidence for any decrease in size following invasion. This invasion‐driven increase in body size sharply contrasts with the pattern observed in many other taxa including plants, mammals and lizards, where invaders frequently exhibit post‐invasion decreases and increases in size. These size changes were not influenced by differences in latitude, sample size or length of time since invasion. Although several mechanisms, may explain the results, none have been demonstrated.
Herbivores and the success of exotic plants: a phylogenetically controlled experiment
Authors: Agrawal Anurag A. , Kotanen Peter M.
Published: July 2003

In a field experiment with 30 locally occurring old‐field plant species grown in a common garden, we found that non‐native plants suffer levels of attack (leaf herbivory) equal to or greater than levels suffered by congeneric native plants. This phylogenetically controlled analysis is in striking contrast to the recent findings from surveys of exotic organisms, and suggests that even if ‘enemy release’ does accompany the invasion process, this may not be an important mechanism of invasion, particularly for plants with close relatives in the recipient flora.
U/Th‐rich bitumen in Archean granites and Palaeoproterozoic metasediments, Rum Jungle Mineral Field, Australia: implications for mineralizing fluids
Authors: McCready A. J. , Stumpfl E. F. , Melcher F.
Published: July 2003

Uranium/thorium (U/Th)‐rich bitumen has been discovered within both Palaeoproterozoic black pelites and the Archean granitic basement of the Rum Jungle Mineral Field, Northern Territory, Australia. Granite‐hosted bitumen occurs as small (up to 400‐µm diameter) discrete individual nodules, which exhibit many morphological similarities to those observed in Phanerozoic siliciclastic rocks. Thorium, the dominant radioelement, occurs primarily as a hydrated Th–Y–Si–P phase. Uranium‐rich inclusions are rare, and correspond to a hydrated U–Th–Y–Si–P phase, identified as coffinite–thorogummite. Metasediment‐hosted bitumen is more variable in morphology, occurring as massive (<2 cm in width) veins that cross‐cut all foliations, as discrete individual nodules or as elongate seams (up to 500 µm in length), interpreted to represent a series of coalesced individual nodules. In all examples, uranium, the dominant radioelement in the metasediment‐hosted bitumen, is present as Th‐poor uraninite, with variable Y2O3contents (up to 3.21 wt.%).

Raman investigation of all types of bitumen indicates that it is a poorly organized carbonaceous matter, which has not been subjected to metamorphism. Consequently, a post‐metamorphic timing for hydrocarbon emplacement can be inferred and a magmatic origin can be precluded.

Potential source rocks for the bitumen are black shales of the Whites Formation (up to 8 wt.% total organic carbon (TOC)) and the Koolpin Formation (approximately 13 wt.% TOC). Post‐metamorphic sericitization of rocks within the Whites Formation is accompanied by a near‐complete removal of organic matter. Alteration was possibly the catalyst for hydrocarbon generation.

The Th–Y–Si–P phase within the granite‐hosted nodules is interpreted to be the result of the alteration of antecedent monazite. During this alteration, U, LREE and P were fractionated and removed, while Th, Y and Si remained immobile, and recombined to form a hydrated Th–Y–Si phase. This pervasive alteration within the basement U/Th‐rich granites is proposed as a genetic model for the formation of uranium deposits in the Rum Jungle Mineral Field and possibly unconformity associated uranium deposits on a global scale.
Geothermal fluids circulation at Caldas do Moledo area, Northern Portugal: geochemical and isotopic signatures
Authors: Marques J. M. , Marques J. Espinha , Carreira P. M. , Graça R. C. , Aires‐Barros L. , Carvalho J. M. , Chaminé H. I. , Borges F. S.
Published: July 2003

A hydrogeological conceptual model of the Caldas do Moledo geothermal site is proposed that shows mixing between geothermal waters and local shallow groundwaters. Stable isotope values of Caldas do Moledo geothermal waters indicate recharge areas located at relatively high altitudes (850–1250 m a.s.l.). The NW–SE Vigo–Régua shear zone plays an important role in fluid recharge and circulation towards the NNE–SSW Régua–Verin fault system, forming a path for ascent of geothermal fluids. The apparent 14C age of geothermal fluids (15.66 ± 2.86 ka BP) was estimated in the total dissolved inorganic carbon (TDIC). Geothermometer calculations indicate that, assuming a conductive temperature gradient of 32°C per kilometer for northern Portugal, the maximum depth of circulation is roughly 1.8 ± 0.4 km. The K, Ca and SO4 concentrations found in some Caldas do Moledo geothermal spring waters show mixing between deep geothermal and shallow groundwater systems. Local shallow groundwaters showing the highest SO4 concentrations were found at low elevation areas, originating from fertilisers and pesticides applied to the Port wine vineyards in the Douro River valley. Geothermal waters from boreholes AC1 and AC2 do not show evidences of direct pollution from the spreading of such agrochemicals.
Abnormally pressured beds as barriers to diffusive solute transport in sedimentary basins
Authors: Wilson Alicia M. , Fenstemaker Thomas , Sharp John M.
Published: July 2003

Diffusion can drive significant solute transport over millions of years, but ancient brines and large salinity gradients are still observed in deep sedimentary basins. Fluid flow within abnormally pressured beds may prevent diffusive transfer over geologically significant periods, if the abnormally pressured bed is surrounded by normally pressured beds. Analytic solutions based on sediment loading and unloading demonstrate that this effect should be considered in beds with a compressibility exceeding 10−8 Pa−1, with a thickness of 100 m or more, or a sedimentation rate exceeding 10−5 m year−1.

Conditions favourable for our model of abnormally pressured beds appear common in sedimentary basins. Large salinity gradients associated with clay beds have previously been attributed to membrane effects, but flow patterns associated with abnormally pressured beds appear more robust in the presence of heterogeneity and discontinuities than membrane effects. Calculations suggest that thick underpressured shales in the Alberta basin may have allowed ancient evaporatively concentrated brines to be preserved beneath a vigorous topography‐driven flow system over the last 60 My. In the Illinois basin, drained overpressured beds may have limited solute transport across the New Albany shale until approximately 250 Ma. It is unlikely, however, that overpressures could have persisted long enough to explain concentration gradients observed in the modern basin. These gradients may instead reflect relatively recent halite dissolution above the New Albany shale.