bluuebunny/crossref_metadata_2025 huggingface dataset:
Fractal analysis of bone texture: a screening tool for stress fracture risk?
Authors: Prouteau S. , Ducher G. , Nanyan P. , Lemineur G. , Benhamou L. , Courteix D.
Published: February 2004

Background  The aim of this study was to identify specific bone characteristics of stress fracture (SF) cases in sportswomen. To date, no tool is able to distinguish individuals who are at risk, limiting preventive measures.

Material and methods  We investigated the skeletal system of sportswomen who did sustain SF in the past (n = 19) and compared it with that of female controls (C) with a similar sporting history but without any fracture history (n = 20).

Bone mass and body composition were measured using dual‐energy X‐ray absorptiometry. Bone micro‐architecture was investigated by calcaneal ultrasound and fractal analysis of calcaneus radiographic images. Oestradiol levels were measured by E.I.A, and IGF‐1 by R.I.A. Menstrual characteristics, nutrient intake, and training were assessed using questionnaires.

Results  The result of the fractal analysis, expressed by the Hmean parameter, was significantly lower in the SF group, reflecting a more complex structure of the trabecular micro‐architectural organization (P < 0·005). Body mass index (BMI) at birth was also found to be lower in the SF cases as compared with their C (P < 0·03).

Multivariate analysis revealed that the fractal parameter Hmean, bone mineral content (BMC) at Ward's triangle and the BMI at birth correctly assigned 84·85% of the female athletes into their respective SF or C groups (P = 0·001).

Conclusion  These results suggest that the fractal parameter and the BMI at birth may be able to identify female athletes most at risk for this overuse bone injury, as their low indexes might reflect a greater skeletal sensitivity.
Brain natriuretic peptide in the prediction of recurrence of angina pectoris
Authors: Takase H. , Toriyama T. , Sugiura T. , Ueda R. , Dohi Y.
Published: February 2004

Background  Circulating levels of brain natriuretic peptide (BNP) provide prognostic information for patients with heart failure, but little is known about its prognostic usefulness in patients with stable angina pectoris. We investigated whether BNP could be used as a marker for the prediction of anginal recurrence after successful treatment.

Design  Brain natriuretic peptide levels of 77 patients with stable angina pectoris were measured at enrolment and after confirmation of successful treatment (i.e. no anginal attack for at least 6 months: chronic phase) with percutaneous transluminal coronary angioplasty and/or conventional medication. Then, we prospectively followed them up for 25·9 ± 1·4 months, with the endpoint being a recurrence of anginal attacks.

Results  An anginal attack recurred in seven patients. In patients without recurrence, BNP levels in the chronic phase (21 ± 12 [median ± median absolute deviation] pg mL−1) were lower than those measured at enrolment (46 ± 25 pg mL−1, P < 0·0001), whereas the levels in patients with recurrence increased during the same period (from 36 ± 16 to 72 ± 42 pg mL−1, P < 0·05). A univariate analysis revealed that the BNP level measured in the chronic phase was the significant predictor of future anginal recurrence. Analysis of the receiver operating characteristic curve indicated that the cutoff level of BNP in the chronic phase was 68 pg mL−1. The Kaplan‐Meier method revealed that the incidence of anginal recurrence was higher in patients with higher (71·4%) than lower levels of BNP (2·9%; P < 0·0001).

Conclusions  Measurement of BNP levels after successful therapy is clinically useful for the prediction of recurrence of anginal attacks in patients with angina pectoris.
Mechanical stretch induces TGF‐β synthesis in hepatic stellate cells
Authors: Sakata R. , Ueno T. , Nakamura T. , Ueno H. , Sata M.
Published: February 2004

Background  It is known that mechanical stress induces extracellular matrix via transforming growth factor‐β (TGF‐β) synthesis in vascular smooth muscle cells. Activated hepatic stellate cells (HSCs) are an important source of TGF‐β in the liver. However, it remains unclear whether mechanical stress induces TGF‐β in HSCs. The Rho small GTP‐binding protein (Rho) has recently emerged as an important regulator of actin and cytoskeleton. We examined whether TGF‐β is expressed in stretched HSCs and whether Rho is involved in stretch‐induced TGF‐β synthesis.

Materials and methods  A cultured human HSC cell line, LI90, was used for this study. Hepatic stellate cells were cyclically stretched using the Flexercell® strain unit. Concentration of TGF‐β in the conditioned medium was estimated by a bioassay using mink lung epithelial cells transfected with a plasminogen activator inhibitor‐1 promoter‐luciferase construct. Transforming growth factor‐β mRNA expression of HSCs was estimated by a reverse‐transcription polymerase chain reaction. Replication‐defective adenoviral vectors expressing a dominant negative type of Rho was utilized to suppress its effect on HSCs.

Results  Transforming growth factor‐β concentration of the conditioned media of stretched HSCs showed time‐dependent increases as compared to nonstretched HSCs from 2 h to 24 h. Transforming growth factor‐β mRNA expression in stretched HSCs was increased compared with that in nonstretched HSCs. Transfection of dominant negative Rho inhibited the stretch‐induced TGF‐β synthesis.

Conclusions  Mechanical stretch enhanced TGF‐β expression on mRNA and protein level in HSCs. Rho was closely related to stretch‐induced TGF‐β synthesis in HSCs.
Toll‐like receptor‐4 Asp299Gly polymorphism does not influence progression of atherosclerosis in patients with familial hypercholesterolaemia
Authors: Netea M. G. , Hijmans A. , Van Wissen S. , Smilde T. J. , Trip M. D. , Kullberg B. J. , De Boo T. , Van der Meer J. W. M. , Kastelein J. J. P. , Stalenhorf A. F. H.
Published: February 2004

Background  Toll‐like receptor‐4 (TLR4) is a major receptor for inflammatory stimuli potentially involved in the pathogenesis of atherosclerosis, such as lipopolysaccharide (LPS) and heat‐shock proteins. The Asp299Gly polymorphism of the TLR4 gene has been associated with a reduced intima‐media thickness (IMT) of the common carotid artery in healthy individuals. We have investigated whether the presence of the Asp299Gly polymorphism in patients with familial hypercholesterolaemia (FH) has a similar protective effect, and whether it influences the effects of HMG‐CoA reductase treatment.

Materials and methods  A cohort of 293 FH patients and 200 healthy volunteers were genotyped for the presence of the Asp299Gly allele using polymerase chain reaction followed by restriction fragment length polymorphism analysis. Intima‐media thickness measurements, inflammatory parameters and the effect of HMG‐CoA reductase inhibitors were compared between the patients with and without Asp299Gly allele.

Results  The Asp299Gly allele was present in 10·6% of the FH patients and 11·0% of the healthy individuals. Whereas the FH patients carrying the Asp299Gly allele displayed a reduced absolute IMT value compared with the FH patients carrying the wild‐type allelle, the difference did not reach statistical significance. In addition, the effect of treatment with HMG‐CoA reductase inhibitors was not influenced by the presence of Asp299Gly allele.

Conclusion  The presence of the Asp299Gly allele of the TLR4 gene does not seem to exert a major influence on the progression of atherosclerosis in patients with FH.
Protein kinase C and extracellular signal regulated kinase are involved in cardiac hypertrophy of rats with progressive renal injury
Authors: Takahashi H. , Takeishi Y. , Miyamoto T. , Shishido T. , Arimoto T. , Konta T. , Miyashita T. , Ito M. , Kubota I.
Published: February 2004

Increased cardiovascular mortality is an unresolved problem in patients with chronic renal failure. Cardiac hypertrophy is observed in the majority of patients with chronic renal failure undergoing haemodialysis. However, the mechanisms, including signal transduction pathways, responsible for cardiac hypertrophy in renal failure remain unknown. We examined the subcellular localization of protein kinase C (PKC) isoforms and phosphorylation activities of 3 mitogen‐activated protein (MAP) kinase families in hypertrophied hearts of progressive renal injury rat model by subtotal nephrectomy (SNx). We also examined the effects of a novel angiotensin II type‐1 receptor antagonist, CS‐866, on the PKC translocation, MAP kinase activity and cardiac hypertrophy in SNx rats. The left ventricle/body weight ratios were significantly larger in SNx rats than in sham rats at 1, 2, and 4 weeks after surgery. The translocation of PKCα and ɛ isoforms to membranous fraction was observed in SNx rat hearts at 1, 2, and 4 weeks after surgery. Activation of extracellular signal regulated kinase (ERK) 1/2, but not p38 MAP kinase and c‐Jun N‐terminal kinase (JNK), was observed at 1 and 2 weeks after surgery. Angiotensin II receptor blockade with CS‐866 (1 mg kg−1 day−1) prevented cardiac hypertrophy, PKC translocation and ERK1/2 activation in SNx rats without significant changes in blood pressure. These data suggest that PKC and ERK1/2 are activated by an angiotensin II receptor‐mediated pathway and might play an important role in the progression of cardiac hypertrophy in renal failure.
Cerebral oximetry in patients with sickle cell disease
Authors: Nahavandi M. , Tavakkoli F. , Hasan S. P. , Wyche M. Q. , Castro O.
Published: February 2004

Background  There is limited information concerning the brain's oxygen supply and demand in patients with sickle cell disease.

Design  We measured near‐infrared spectroscopy of brain oxygenation in 27 patients with sickle cell disease regardless of vaso‐occlusive crisis, 14 normal healthy controls, and five anaemic patients without sickle cell disease. We also measured pre‐ and post‐transfusion cerebral oximetry in 14 additional sickle cell disease patients who were on transfusion programmes.

Results  The mean cerebral oxygen saturation in the combined steady‐state and vaso‐occlusive crisis population was found to be significantly lower than that in the controls and in anaemic patients without sickle cell disease (47·7% vs. 61·3%, 59·8%, P < 0·0001). Cerebral oxygen saturation failed to correlate with the haemoglobin concentration (r = 0·51, P > 0·5). However, cerebral oxygen saturation increased from 40·4% to 49·6% (P = 0·01) and correlated significantly with the haemoglobin level (r = 0·553, P = 0·003) in 14 subjects studied before and after transfusions. In seven subjects who received simple transfusions, cerebral oxygen saturation correlated strongly and positively with the haemoglobin level (r = 0·811, P = 0·001) and with percent normal haemoglobin (r = 0·786, P = 0·002), and negatively with abnormal sickle haemoglobin (r = −0·775, P = 0·003). None of these correlations was found to be statistically significant in the seven subjects given exchange transfusions. Cerebral oxygen saturation measured in the sickle cell disease subjects after transfusions was still significantly lower than in the anaemic subjects without sickle cell disease and in the normal controls (49·6% vs. 59·8% and 61·3%, P = 0·001).

Conclusions  We found that patients with sickle cell disease have subnormal values of cerebral oxygen saturation. Red cell transfusions significantly increased the brain oxygenation in these patients. Cerebral oximetry may be a useful, noninvasive method for assessing the effect of circulating normal red cells in sickle cell patients after transfusions.
Identification of Neurones in the Female Rat Hypothalamus That Express Oestrogen Receptor‐Alpha and Vesicular Glutamate Transporter‐2
Authors: Eyigor O. , Lin W. , Jennes L.
Published: February 2004

Oestrogen exerts its effects in the brain by binding to and activating two members of the nuclear receptor family, oestrogen receptor (ER)‐α and ER‐β. Evidence suggests that oestrogen‐receptive neurones participate in the generation of reproductive behaviours and that they convey the oestrogen message to gonadotropin‐releasing hormone (GnRH) neurones. The aim of the present study was to identify the neurochemical phenotype of a subset of oestrogen receptor‐expressing neurones. To this aim, we focused on the glutamate neuronal system, which is one of the most important stimulators of GnRH synthesis and release. We used the presence of vesicular glutamate transporter‐2 (VGLUT2) mRNA as a specific marker to identify glutamate neurones and employed dual in situ hybridization to localize ERα mRNA‐(35S‐labelling) and VGLUT2 mRNA‐(digoxigenin‐labelling) expressing neurones within the hypothalamus. The results show that the overall distribution of VGLUT2 mRNA and ERα mRNA are consistent with previous data in the literature. Dual‐labelled neurones were localized in the ventrolateral part of the ventromedial nucleus where 81.3 ± 3.4% of the ERα mRNA containing neurones expressed VGLUT2 mRNA, in the anteroventral periventricular nucleus (30% colocalization) and in the medial preoptic nucleus (19% colocalization). Only 4.4% of the ERα expressing neurones in the arcuate nucleus contained VGLUT2 mRNA. These findings reveal that certain subpopulations of oestrogen‐receptive neurones are glutamatergic in select hypothalamic areas that are known to regulate reproductive behaviour and GnRH neurones in the female rat. Thus, the oestrogen signal could be propagated through glutamate neurones to distant sites and influence the activity of the postsynaptic neurones.
Regulation of Galanin‐Like Peptide Gene Expression By Pituitary Hormones and Their Downstream Targets
Authors: Cunnningham M. J. , Krasnow S. M. , Gevers E. F. , Chen P. , Thompson C. K. , Robinson I. C. A. F. , Smith M. S. , Clifton D. K. , Steiner R. A.
Published: February 2004

Galanin‐like peptide (GALP) mRNA is expressed in neurones of the hypothalamic arcuate nucleus and within pituicytes in the neurohypophysis. Several neuropeptides that are expressed in the arcuate nucleus participate in the neuroendocrine regulation of pituitary hormone secretion. Our objective was to determine the extent to which GALP might be a target for regulation by pituitary hormones or their downstream targets in the rat. The expression of GALP mRNA in the arcuate nucleus was reduced by hypophysectomy as determined by in situ hybridization. However, this did not appear to be attributable to the loss of either gonadal or adrenal steroids because castrated, ovariectomized and adrenalectomized rats had GALP mRNA expression that was indistinguishable from their respective controls. Next, we investigated the effects of growth hormone deficiency on GALP mRNA expression by studying dwarf rats and found that GALP gene expression was not different between dwarf rats and controls. We found that thyroidectomy led to a significant reduction in GALP mRNA expression compared to intact controls, and thyroidectomized rats implanted with thyroxine pellets had GALP mRNA expression that was similar to intact controls. Thus, the reduction of GALP mRNA expression seen in hypophysectomized animals may reflect, in part, a selective loss of thyroid hormone. We also found that the expression of GALP mRNA was increased in the neurohypophysis of lactating rats compared to nonlactating rats, whereas GALP mRNA expression in the arcuate nucleus was unaffected by lactation. This suggests that the induction of GALP gene expression in pituicytes is physiologically associated with activation of oxytocin and vasopressin secretion during lactation.
Expression of the Arginine Vasopressin Gene in Response to Salt Loading in Oxytocin Gene Knockout Mice
Authors: Ozaki Y. , Nomura M. , Saito J. , Luedke C. E. , Muglia L. J. , Matsumoto T. , Ogawa S. , Ueta Y. , Pfaff D. W.
Published: February 2004

Accumulating evidence suggests that both oxytocin and arginine vasopressin (AVP) are vital components in the regulation of body fluid balance. However, the physiological role of oxytocin and possible cooperative interactions between oxytocin and AVP in sodium balance remain obscure, even though recent studies using oxytocin knockout (OTKO) mice suggested that oxytocin may contribute to the regulation of salt appetite. In the present study, we examined the effects of salt loading (drinking 2% NaCl for 5 days) on the expression of the AVP gene in the paraventricular (PVN) and supraoptic nuclei (SON) of wild‐type, OTKO and heterozygous littermates using in situ hybridization histochemistry. In addition, the effects of salt loading on the expression of the oxytocin gene were also examined in wild‐type and heterozygous mice. Under the non salt‐loaded condition, the levels of AVP mRNA in the PVN and SON of OTKO mice were significantly decreased compared to those in wild‐type mice. Nevertheless, the up‐regulation of the expression of the AVP gene in response to salt loading was preserved in OTKO mice. The degree of the up‐regulation in OTKO mice tended to be greater compared to those in wild‐type mice, suggesting compensatory up‐regulation of the expression of the AVP gene in OTKO mice after salt loading. The basal levels of oxytocin mRNA in the PVN and SON of heterozygous mice were significantly lower than those in wild‐type mice. Salt loading caused an increase of oxytocin mRNA levels in the PVN and SON of both wild‐type and heterozygous mice. The ratios of increase of oxytocin mRNA levels were very similar between wild‐type and heterozygous mice, suggesting that the single remaining oxytocin gene in heterozygous mice responds normally to an osmotic cue. Finally, salt loading tended to increase the serum concentration of sodium regardless of genotype, and there were no genotype differences in both the control and salt‐loaded groups. These results suggest ways in which oxytocin may play a cooperative role together with AVP in the regulation of sodium balance.
Dual Excitatory and Inhibitory Effects of Stimulation of Intrinsic Innervation of the Anterior Pituitary on Adrenocorticotropic Hormone Release in the Rat
Authors: Gao L.‐Z. , Ju G.
Published: February 2004

The gland cells of the mammalian anterior pituitary are innervated by substantial amounts of nerve fibres, and there is evidence that the nerve fibres are functionally active. In the rat, the nerve fibres make typical excitatory synapses with corticotropes. The physiological significance of this synaptic relationship was investigated in the present study. The anterior pituitary of the rat was sliced and stimulated with electrical field in a chamber. The perfusate was continuously collected and immunoradioassayed for adrenocorticotropic hormone (ACTH). When the gland slices were stimulated at a high frequency of 10 Hz, there was a significant inhibition of ACTH secretion. Stimulation at a low frequency of 2 Hz resulted in a quick and transient excitation of ACTH release. The results indicate that stimulation of the nerve fibres in the anterior pituitary has dual excitatory and inhibitory effects on ACTH secretion.
Reduced Progesterone Metabolites Protect Rat Hippocampal Neurones From Kainic Acid Excitotoxicity In Vivo
Authors: Ciriza I. , Azcoitia I. , Garcia‐Segura L. M.
Published: February 2004

The ovarian hormone progesterone is neuroprotective in some animal models of neurodegeneration. Progesterone actions in the brain may partly be mediated by the locally produced metabolites 5α‐dihydroprogesterone and 3α,5α‐tetrahydroprogesterone. The neuroprotective effects of these two metabolites of progesterone were assessed in this study. Ovariectomized Wistar rats were injected with kainic acid, to induce excitotoxic neuronal death in the hippocampus, and with different doses of 5α‐dihydroprogesterone and 3α,5α‐tetrahydroprogesterone. The number of surviving neurones in the hilus of the dentate gyrus of the hippocampus was assessed with the optical disector method. The administration of kainic acid resulted in a significant decrease in the number of hilar neurones and in the induction of vimentin expression in reactive astrocytes, a sign of neural damage. Low doses of 5α‐dihydroprogesterone (0.25 and 0.5 mg/kg body weight, b.w.) prevented the loss of hilar neurones and the appearance of vimentin immunoreactivity in astrocytes. Higher doses (1–2 mg/kg b.w.) were not neuroprotective. By contrast, low doses of 3α,5α‐tetrahydroprogesterone (0.25–1 mg/kg b.w.) were unable to protect the hilus from kainic acid while higher doses (2–4 mg/kg b.w.) were protective. The different optimal neuroprotective doses of 5α‐dihydroprogesterone and 3α,5α‐tetrahydroprogesterone suggest that these two steroids may protect neurones using different mechanisms. The neuroprotective effects of 3α,5α‐tetrahydroprogesterone may be exerted by the inhibition of neuronal activity via the GABAA receptor. This latter possibility is supported by the observation that 3β,5α‐tetrahydroprogesterone, an isomer of 3α,5α‐tetrahydroprogesterone that does not bind to GABAA receptor, was not neuroprotective. In summary, our findings suggest that progesterone neuroprotective effects may be, at least in part, mediated by its reduced metabolites 5α‐dihydroprogesterone and 3α,5α‐tetrahydroprogesterone.
Altered Expression of p53 mRNA in the Brain and Pituitary During Repeated Immobilization Stress: Negative Correlation with Glucocorticoid Receptor mRNA Levels
Authors: Nishimura K. , Makino S. , Tanaka Y. , Kaneda T. , Hashimoto K.
Published: February 2004

In our previous study, apparent reduction of glucocorticoid receptor (GR) mRNA was seen in the hippocampus and the hypothalamic paraventricular nucleus (PVN) during repeated immobilization (IMO) stress, but not following starvation. Our laboratory has also shown that the sp1 activates, whereas tumour suppressor p53 represses the promoter activity of GR gene. In an attempt to reveal the possibility that transcription factors such as sp1 and/or p53 are involved in the regulation of GR mRNA expression in the hippocampus and in the PVN in vivo, we examined the expression of GR mRNA, p53 mRNA, and sp1 mRNA in the hippocampus and in the PVN during repeated IMO and following starvation. In addition, the expression of these mRNAs was examined in the anterior pituitary, another GR‐rich area. GR mRNA in all subfields of the hippocampus was robustly decreased, while GR mRNA in the anterior pituitary was increased, 24 h following 4 × IMO (2 h daily, for 4 consecutive days) and immediately after 5 × IMO. GR mRNA in the PVN was significantly decreased immediately after 5 × IMO, but not at 24 h after 4 × IMO. Conversely, p53 mRNA in the PVN and hippocampus was increased, whereas p53 mRNA in the anterior pituitary was decreased, 24 h following 4 × IMO and immediately after 5 × IMO. Sp1 mRNA was unchanged in all areas examined following repeated IMO. Following 4 days of starvation, neither GR mRNA, p53 mRNA nor sp1 mRNA showed any changes in the PVN and the hippocampus, except there was a minor decrease in GR mRNA in CA1‐2. In the anterior pituitary, 4 days of starvation induced a minor, but significant increase in GR mRNA, whereas it decreased p53 mRNA. Overall, regression analyses revealed a negative correlation between GR mRNA levels and p53 mRNA levels in CA1‐2 and dentate gyrus of the hippocampus and in the anterior pituitary. GR mRNA in the PVN also showed a tendency towards the negative correlation with p53 mRNA levels. The results raise the possibility that p53 negatively regulates GR mRNA expression in the PVN, the hippocampus and the anterior pituitary during repeated immobilization stress.
Circadian Rhythm of Melatonin Release From the Photoreceptive Pineal Organ of a Teleost, Ayu (Plecoglossus altivelis) in Flow‐Thorough Culture
Authors: Iigo M. , Fujimoto Y. , Gunji‐Suzuki M. , Yokosuka M. , Hara M. , Ohtani‐Kaneko R. , Tabata M. , Aida K. , Hirata K.
Published: February 2004

In the present study, we tested whether the pineal organ of ayu (Plecoglossus altivelis), an osmerid teleost close relative of salmonids, harbours a circadian oscillator regulating rhythmic melatonin release using flow‐through culture. The pineal organ maintained under light/dark cycles released melatonin in a rhythmic fashion with high levels during the dark phase. A circadian rhythm of melatonin release persisted in constant darkness for at least four cycles. Characteristics of the circadian rhythm (free‐running period, phase and amplitude) exhibited small variations among cultures when the data was normalized, indicating that this system is sufficient for the analysis of the circadian rhythm both at qualitative and quantitative levels. Six‐hour extension of the light phase from the normal onset time of the dark phase or exposure to constant light for 36 or 48 h before transfer to constant darkness significantly inhibited melatonin release. Phase shifts in the circadian rhythm of melatonin release were also observed. Thus, the ayu pineal organ contains all the three essential components of the circadian system (a circadian clock, the photoreceptor responsible for photic entrainment of the clock, and melatonin generating system as an output pathway). This system should provide a useful model for analysing the physiological and molecular basis of the vertebrate circadian system. In addition, further comparative studies using salmonids and related species including ayu will provide some insight into the evolution of the roles of the pineal organ in the vertebrate circadian system.
The role of N‐linked glycosylation in the protection of human and bovine lactoferrin against tryptic proteolysis
Authors: van Veen Harrie A. , Geerts Marlieke E. J. , van Berkel Patrick H. C. , Nuijens Jan H.
Published: February 2004

Lactoferrin (LF) is an iron‐binding glycoprotein of the innate host defence system. To elucidate the role of N‐linked glycosylation in protection of LF against proteolysis, we compared the tryptic susceptibility of human LF (hLF) variants from human milk, expressed in human 293(S) cells or in the milk of transgenic mice and cows. The analysis revealed that recombinant hLF (rhLF) with mutations Ile130→Thr and Gly404→Cys was about twofold more susceptible than glycosylated and unglycosylated variants with the naturally occurring Ile130 and Gly404. Hence, N‐linked glycosylation is not involved in protection of hLF against tryptic proteolysis. Apparently, the previously reported protection by N‐linked glycosylation of hLF [van Berkel, P.H.C., Geerts, M.E.J., van Veen, H.A., Kooiman, P.M., Pieper, F., de Boer, H.A. & Nuijens, J.H. (1995) Biochem. J. 312, 107–114] is restricted to rhLF containing the Thr130 and Cys404. Comparison of the tryptic proteolysis of hLF and bovine LF (bLF) revealed that hLF is about 100‐fold more resistant than bLF. Glycosylation variants A and B of bLF differed by about 10‐fold in susceptibility to trypsin. This difference is due to glycosylation at Asn281 in bLF‐A. Hence, glycosylation at Asn281 protects bLF against cleavage by trypsin at Lys282.
Cloning and characterization of two distinct isoforms of rainbow trout heat shock factor 1
Authors: Ojima Nobuhiko , Yamashita Michiaki
Published: February 2004

To elucidate the molecular mechanism underlying the heat shock response in cold‐water fish species, genes encoding heat shock transcription factors (HSFs) were cloned from RTG‐2 cells of the rainbow trout Oncorhynchus mykiss. Consequently, two distinct HSF1 genes, named HSF1a and HSF1b, were identified. The predicted amino acid sequence of HSF1a shows 86.4% identity to that of HSF1b. The two proteins contained the general structural motifs of HSF1, i.e. a DNA‐binding domain, hydrophobic heptad repeats and nuclear localization signals. Southern blot analysis showed that each HSF1 is encoded by a distinct gene. The two HSF1 mRNAs were coexpressed in unstressed rainbow trout RTG‐2 cells and in various tissues. In an electrophoretic mobility shift assay, each in vitro translated HSF1 bound to the heat shock element. Chemical cross‐linking and immunoprecipitation analysis showed that HSF1a and HSF1b form heterotrimers as well as homotrimers. Taken together, these results demonstrate that in rainbow trout cells there are two distinct HSF1 isoforms that can form heterotrimers, suggesting that a unique molecular mechanism underlies the stress response in tetraploid and/or cold‐water fish species.
UDPgalactose 4‐epimerase from Saccharomyces cerevisiae
Authors: Majumdar Siddhartha , Ghatak Jhuma , Mukherji Sucheta , Bhattacharjee Hiranmoy , Bhaduri Amar
Published: February 2004

UDPgalactose 4‐epimerase (epimerase) catalyzes the reversible conversion between UDPgalactose and UDPglucose and is an important enzyme of the galactose metabolic pathway. The Saccharomyces cerevisiae epimerase encoded by the GAL10 gene is about twice the size of either the bacterial or human protein. Sequence analysis indicates that the yeast epimerase has an N‐terminal domain (residues 1–377) that shows significant similarity with Escherichia coli and human UDPgalactose 4‐epimerase, and a C‐terminal domain (residues 378–699), which shows extensive identity to either the bacterial or human aldose 1‐epimerase (mutarotase). The S. cerevisiae epimerase was purified to > 95% homogeneity by sequential chromatography on DEAE‐Sephacel and Resource‐Q columns. Purified epimerase preparations showed mutarotase activity and could convert either α‐d‐glucose or α‐d‐galactose to their β‐anomers. Induction of cells with galactose led to simultaneous enhancement of both epimerase and mutarotase activities. Size exclusion chromatography experiments confirmed that the mutarotase activity is an intrinsic property of the yeast epimerase and not due to a copurifying endogenous mutarotase. When the purified protein was treated with 5′‐UMP and l‐arabinose, epimerase activity was completely lost but the mutarotase activity remained unaffected. These results demonstrate that the S. cerevisiae UDPgalactose 4‐epimerase is a bifunctional enzyme with aldose 1‐epimerase activity. The active sites for these two enzymatic activities are located in different regions of the epimerase holoenzyme.
Non‐specific depolymerization of chitosan by pronase and characterization of the resultant products
Authors: Kumar Acharya B. Vishu , Gowda Lalitha R. , Tharanathan Rudrapatnam N.
Published: February 2004

Pronase (type XXV serine protease from Streptomyces griseus) efficiently depolymerizes chitosan, a linear β→1,4‐linked polysaccharide of 2‐amino‐deoxyglucose and 2‐amino‐2‐N‐acetylamino‐d‐glucose, to low‐molecular weight chitosans (LMWC), chito‐oligomers (degree of polymerization, 2–6) and monomer. The maximum depolymerization occurred at pH 3.5 and 37 °C, and the reaction obeyed Michaelis–Menten kinetics with a Km of 5.21 mg·mL−1 and Vmax of 138.55 nmoles·min−1·mg−1. The molecular mass of the major product, LMWC, varied between 9.0 ± 0.5 kDa depending on the reaction time. Scanning electron microscopy of LMWC showed an approximately eightfold decrease in particle size and characterization by infrared spectroscopy, circular dichroism, X‐ray diffractometry and 13C‐NMR revealed them to possess a lower degree of acetylation, hydration and crystallinity compared to chitosan. Chitosanolysis by pronase is an alternative and inexpensive method to produce a variety of chitosan degradation products that have wide and varied biofunctionalities.
ICAM‐1 expression is highly NF‐κB‐dependent in A549 cells
Authors: Holden Neil S. , Catley Matthew C. , Cambridge Lisa M. , Barnes Peter J. , Newton Robert
Published: February 2004

The transcription factor nuclear factor κB (NF‐κB) is an activator of multiple cytokines, chemokines and adhesion molecules, which are important in inflammatory diseases such as asthma, and is consequently considered as an attractive therapeutic target. In the present study, a constitutively active dominant version of IκBα, IκBαDN, was introduced into A549 pulmonary cells by adenovirus‐mediated delivery. The dominant IκB, but not a null viral vector, prevented the induction of NF‐κB‐dependent transcription by both tumor necrosis factor α (TNFα) and interleukin‐1β (IL‐1β). Similarly, both TNFα and IL‐1β strongly induced mRNA and protein expression of intercellular adhesion molecule (ICAM)‐1 and in each case this was prevented by adenovirus expressing the dominant IκB, but not by the null virus, thereby establishing ICAM‐1 as an NF‐κB‐dependent gene. Numerous studies have suggested key roles for the p38 and extracellular regulated kinase (ERK) mitogen‐activated protein kinase (MAPK) cascades in the activation and transactivation of NF‐κB. We show here that SB203580, a selective inhibitor of the p38 MAPK, and PD098059 and UO126, both selective inhibitors of the ERK MAPK cascade, have no effect on TNFα or IL‐1β‐induced translocation and DNA binding of NF‐κB. Furthermore, these inhibitors showed no pharmacologically relevant effect on NF‐κB‐dependent transcription nor was there any effect on expression of ICAM‐1. Taken together these data highlight the potential use of inhibition of the NF‐κB signalling pathway in pulmonary inflammatory diseases and suggest that inhibitors of the p38 and ERK MAPK pathways may be of lesser effect.
FRET evidence for a conformational change in TFIIB upon TBP‐DNA binding
Authors: Zheng Le , Hoeflich Klaus P. , Elsby Laura M. , Ghosh Mahua , Roberts Stefan G. E. , Ikura Mitsuhiko
Published: February 2004

As a critical step of the preinitiation complex assembly in transcription, the general transcription factor TFIIB forms a complex with the TATA‐box binding protein (TBP) bound to a promoter element. Transcriptional activators such as the herpes simplex virus VP16 facilitate this complex formation through conformational activation of TFIIB, a focal molecule of transcriptional initiation and activation. Here, we used fluorescence resonance energy transfer to investigate conformational states of human TFIIB fused to enhanced cyan fluorescent protein and enhanced yellow fluorescent protein at its N‐ and C‐terminus, respectively. A significant reduction in fluorescence resonance energy transfer ratio was observed when this fusion protein, hereafter named CYIIB, was mixed with promoter‐loaded TBP. The rate for the TFIIB–TBP–DNA complex formation is accelerated drastically by GAL4‐VP16 and is also dependent on the type of promoter sequences. These results provide compelling evidence for a ‘closed‐to‐open’ conformational change of TFIIB upon binding to the TBP–DNA complex, which probably involves alternation of the spatial orientation between the N‐terminal zinc ribbon domain and the C‐terminal conserved core domain responsible for direct interactions with TBP and a DNA element.
Selective release and function of one of the two FMN groups in the cytoplasmic NAD+‐reducing [NiFe]‐hydrogenase from Ralstonia eutropha
Authors: van der Linden Eddy , Faber Bart W. , Bleijlevens Boris , Burgdorf Tanja , Bernhard Michael , Friedrich Bärbel , Albracht Simon P. J.
Published: February 2004

The soluble, cytoplasmic NAD+‐reducing [NiFe]‐hydrogenase from Ralstonia eutropha is a heterotetrameric enzyme (HoxFUYH) and contains two FMN groups. The purified oxidized enzyme is inactive in the H2‐NAD+ reaction, but can be activated by catalytic amounts of NADH. It was discovered that one of the FMN groups (FMN‐a) is selectively released upon prolonged reduction of the enzyme with NADH. During this process, the enzyme maintained its tetrameric form, with one FMN group (FMN‐b) firmly bound, but it lost its physiological activity – the reduction of NAD+ by H2. This activity could be reconstituted by the addition of excess FMN to the reduced enzyme. The rate of reduction of benzyl viologen by H2 was not dependent on the presence of FMN‐a. Enzyme devoid of FMN‐a could not be activated by NADH. As NADH‐dehydrogenase activity was not dependent on the presence of FMN‐a, and because FMN‐b did not dissociate from the reduced enzyme, we conclude that FMN‐b is functional in the NADH‐dehydrogenase activity catalyzed by the HoxFU dimer. The possible function of FMN‐a as a hydride acceptor in the hydrogenase reaction catalyzed by the HoxHY dimer is discussed.
Nonlysine‐analog plasminogen modulators promote autoproteolytic generation of plasmin(ogen) fragments with angiostatin‐like activity
Authors: Ohyama Shigeki , Harada Tomotaka , Chikanishi Toshihiro , Miura Yutaka , Hasumi Keiji
Published: February 2004

We recently discovered several nonlysine‐analog conformational modulators for plasminogen. These include SMTP‐6, thioplabin B and complestatin that are low molecular mass compounds of microbial origin. Unlike lysine‐analog modulators, which increase plasminogen activation but inhibit its binding to fibrin, the nonlysine‐analog modulators enhance both activation and fibrin binding of plasminogen. Here we show that some nonlysine‐analog modulators promote autoproteolytic generation of plasmin(ogen) derivatives with its catalytic domain undergoing extensive fragmentation (PMDs), which have angiostatin‐like anti‐endothelial activity. The enhancement of urokinase‐catalyzed plasminogen activation by SMTP‐6 was followed by rapid inactivation of plasmin due to its degradation mainly in the catalytic domain, yielding PMD with a molecular mass ranging from 68 to 77 kDa. PMD generation was observed when plasmin alone was treated with SMTP‐6 and was inhibited by the plasmin inhibitor aprotinin, indicating an autoproteolytic mechanism in PMD generation. Thioplabin B and complestatin, two other nonlysine‐analog modulators, were also active in producing similar PMDs, whereas the lysine analog 6‐aminohexanoic acid was inactive while it enhanced plasminogen activation. Peptide sequencing and mass spectrometric analyses suggested that plasmin fragmentation was due to cleavage at Lys615‐Val616, Lys651‐Leu652, Lys661‐Val662, Lys698‐Glu699, Lys708‐Val709 and several other sites mostly in the catalytic domain. PMD was inhibitory to proliferation, migration and tube formation of endothelial cells at concentrations of 0.3–10 µg·mL−1. These results suggest a possible application of nonlysine‐analog modulators in the treatment of cancer through the enhancement of endogenous plasmin(ogen) fragment formation.
Identification of a gene encoding Lon protease from Brevibacillus thermoruber WR‐249 and biochemical characterization of its thermostable recombinant enzyme
Authors: Lee Tsay Alan Y.‐L., San‐San , Chen Mao‐Yen , Wu Shih‐Hsiung
Published: February 2004

A gene encoding thermostable Lon protease from Brevibacillus thermoruber WR‐249 was cloned and characterized. The Br. thermoruber Lon gene (Bt‐lon) encodes an 88 kDa protein characterized by an N‐terminal domain, a central ATPase domain which includes an SSD (sensor‐ and substrate‐discrimination) domain, and a C‐terminal protease domain. The Bt‐lon is a heat‐inducible gene and may be controlled under a putative Bacillus subtilisσA‐dependent promoter, but in the absence of CIRCE (controlling inverted repeat of chaperone expression). Bt‐lon was expressed in Escherichia coli, and its protein product was purified. The native recombinant Br. thermoruber Lon protease (Bt‐Lon) displayed a hexameric structure. The optimal temperature of ATPase activity for Bt‐Lon was 70 °C, and the optimal temperature of peptidase and DNA‐binding activities was 50 °C. This implies that the functions of Lon protease in thermophilic bacteria may be switched, depending on temperature, to regulate their physiological needs. The peptidase activity of Bt‐Lon increases substantially in the presence of ATP. Furthermore, the substrate specificity of Bt‐Lon is different from that of E. coli Lon in using fluorogenic peptides as substrates. Notably, the Bt‐Lon protein shows chaperone‐like activity by preventing aggregation of denatured insulin B‐chain in a dose‐dependent and ATP‐independent manner. In thermal denaturation experiments, Bt‐Lon was found to display an indicator of thermostability value, Tm of 71.5 °C. Sequence comparison with mesophilic Lon proteases shows differences in the rigidity, electrostatic interactions, and hydrogen bonding of Bt‐Lon relevant to thermostability.
Acharan sulfate, the new glycosaminoglycan from Achatina fulica Bowdich 1822
Authors: Vieira Tuane C. R. G. , Costa‐Filho Adilson , Salgado Norma C. , Allodi Silvana , Valente Ana‐Paula , Nasciutti Luiz E. , Silva Luiz‐Claudio F.
Published: February 2004

Acharan sulfate, a recently discovered glycosaminoglycan isolated from Achatina fulica, has a major disaccharide repeating unit of →4)‐2‐acetyl,2‐deoxy‐α‐d‐glucopyranose(1→4)‐2‐sulfo‐α‐l‐idopyranosyluronic acid (1→, making it structurally related to both heparin and heparan sulfate. It has been suggested that this glycosaminoglycan is polydisperse, with an average molecular mass of 29 kDa and known minor disaccharide sequence variants containing unsulfated iduronic acid. Acharan sulfate was found to be located in the body of this species using alcian blue staining and it was suggested to be the main constituent of the mucus. In the present work, we provide further information on the structure and compartmental distribution of acharan sulfate in the snail body. Different populations of acharan sulfate presenting charge and/or molecular mass heterogeneities were isolated from the whole body, as well as from mucus and from the organic shell matrix. A minor glycosaminoglycan fraction susceptible to degradation by nitrous acid was also purified from the snail body, suggesting the presence of N‐sulfated glycosaminoglycan molecules. In addition, we demonstrate the in vivo metabolic labeling of acharan sulfate in the snail body after a meal supplemented with [35S]free sulfate. This simple approach might be applied to the study of acharan sulfate biosynthesis. Finally, we developed histochemical assays to localize acharan sulfate in the snail body by metachromatic staining and by histoautoradiography following metabolic radiolabeling with [35S]sulfate. Our results show that acharan sulfate is widely distributed among several organs.
Vertical‐scanning mutagenesis of amino acids in a model N‐myristoylation motif reveals the major amino‐terminal sequence requirements for protein N‐myristoylation
Authors: Utsumi Toshihiko , Nakano Kengo , Funakoshi Takeshi , Kayano Yoshiyuki , Nakao Sayaka , Sakurai Nagisa , Iwata Hiroyuki , Ishisaka Rumi
Published: February 2004

In order to determine the amino‐terminal sequence requirements for protein N‐myristoylation, site‐directed mutagenesis of the N‐terminal region was performed using tumor necrosis factor (TNF) mutants as model substrate proteins. Subsequently, the susceptibility of these mutants to protein N‐myristoylation was evaluated by metabolic labeling in an in vitro translation system using rabbit reticulocyte lysate. A TNF mutant having the sequence MGAAAAAAAA at its N‐terminus was used as the starting sequence to identify elements critical for protein N‐myristoylation. Sequential vertical‐scanning mutagenesis of amino acids at a distinct position in this model N‐terminal sequence revealed the major sequence requirements for protein N‐myristoylation: the combination of amino acids at position 3 and 6 constitutes a major determinant for the susceptibility to protein N‐myristoylation. When Ser was located at position 6, 11 amino acids (Gly, Ala, Ser, Cys, Thr, Val, Asn, Leu, Ile, Gln, His) were permitted at position 3 to direct efficient protein N‐myristoylation. In this case, the presence of Lys at position 7 was found to affect the amino acid requirement at position 3 and Lys became permitted at this position. When Ser was not located at position 6, only 3 amino acids (Ala, Asn, Gln) were permitted at position 3 to direct efficient protein N‐myristoylation. The amino acid requirements found in this study were fully consistent with the N‐terminal sequence of 78 N‐myristoylated proteins in which N‐myristoylation was experimentally verified. These observations strongly indicate that the combination of amino acids at position 3, 6 and 7 is a major determinant for protein N‐myristoylation.
Effects of population spatial structure on the quantity and quality of seeds set byPrimula sieboldii(Primulaceae)
Authors: WATANABE ATSUKO , GOKA KOUICHI , WASHITANI IZUMI
Published: February 2004

Effects of spatial population structure on the reproductive success ofPrimula sieboldiiE. Morren, an endangered heterostylous perennial, were studied through the analysis of quantity and quality of seeds in a natural habitat in central Japan. Seed set of individual flowering ramets varied significantly depending on the degrees of isolation from their potential mates. The flowering ramets, which have the opposite‐morph ramets within 5 m, set significantly higher mean seeds per flower (approx. 15) than that of more isolated ramets (approx. 5). The former produced a considerably variable number (0–100) of seeds per flower, while the latter set more or less uniform number of seeds per flower. Viability of seeds from the former ramets was also variable with significantly higher mean viability percentage than the latter. Early seedling survival was also significantly different between the maternal isolation classes, while survival and growth of the established seedlings in a greenhouse did not differ significantly between these classes. The level of allozyme heterozygosity of the surviving progenies was not significantly different between the classes differing in maternal isolation. The observation suggests that the isolated maternal plants produced fewer and less viable seeds, which may be a result of strong inbreeding depression that manifests in early life stages of the progenies. By the stage of seedling establishment, therefore, less fit homozygotes are purged while fitter heterozygotes are allowed to survive irrespective of maternal isolation.
Oxylobium/Gastrolobium(Fabaceae : Mirbelieae) conundrum: further studies using molecular data and a reappraisal of morphological characters
Authors: CHANDLER GREGORY T. , BAYER RANDALL J. , GILMORE SIMON R.
Published: February 2004

A molecular phylogeny ofOxylobium,Gastrolobiumand related genera, includingBrachysema,Jansonia,NemciaandPodolobium,is presented. The study was conducted using five molecular regions and 48 taxa, utilizing chloroplast DNA (thetrnK 5′ intron,psbA/trnH intergenic spacer,trnL intron and adjacenttrnL/trnF intergenic spacer) and nuclear ribosomal DNA (ETS and ITS) markers.Oxylobiumis shown to be polyphyletic, whileGastrolobiumis paraphyletic, containing within it the generaBrachysema,JansoniaandNemcia, as well asOxylobium lineare. This concurs with a previous molecular analysis and the results of the two analyses are compared. Morphological traits, such as subtending bracts, ovule number and fluoroacetate content, are shown to be homoplastic and suites of characters for defining the different genera using different morphological characters are presented. Our results support a recent monograph that expandsGastrolobiumto includeBrachysema,Jansonia,NemciaandOxylobium lineare. This revision leavesOxylobiumandPodolobiumoccurring exclusively in eastern Australia, whileGastrolobiumoccurs almost exclusively in south‐western Australia, with only two species,G. brevipesandG. grandiflorum, occurring outside this area.
Effects of nutrient availability on the production of pentaynene, a secondary compound related to defense, in Rudbeckia hirta
Authors: ALMEIDA‐CORTEZ JARCILENE , SHIPLEY BILL , ARNASON JOHN THOR
Published: February 2004

This study focused on the effect of different availabilities of mineral resources in hydroponic culture on the production of polyacetylenic defences of Rudbeckia hirta L. Specifically, we investigated if there was any difference concerning the amount of pentaynene, measured with high performance liquid chromatography (HPLC), produced in flowers of plants grown at high light intensity (500 µmol m−2 s−1 photosynthetic photon flux density) and two levels of mineral resources (full strength and 1/5 dilution) in a hydroponic solution. A working hypothesis was that the concentration of secondary compounds would increase with decreasing nutrient supply, in accordance with the carbon:nutrient balance hypothesis. The amount of pentaynene produced in the flowers of R. hirta decreased with increasing flower age but increased significantly when the plant was subjected to a reduced nutrient availability. The decrease in pentaynene concentrations with flower age was also less pronounced in the nutrient‐stressed plants.