bluuebunny/crossref_metadata_2025 huggingface dataset:
Visualization of cell cycling by an improvement in slice culture methods
Authors: Miyata Takaki , Kawaguchi Ayano , Saito Kanako , Kuramochi Hiroshi , Ogawa Masaharu
Published: October 2002

Slice culture combined with the use of fluorescent dyes and/or the introduction of fluorescent protein genes provides live and three‐dimensional information on cytogenetic and histogenetic events at the level of the individual cell. Using slices prepared from midembryonic mouse cerebral wall tissue upon which fine DiI crystals were placed on the pial or ventricular surface, we recently found that dividing progenitor cells do not lose their pia‐connected (basal) processes and that the processes are inherited by daughter cells, including neurons (Miyata et al. [2001] Neuron 31:727–741). To understand more fully the biological significance of this inheritance process, the fate of each daughter cell should be monitored over a culture period extended long enough to allow a neuron to migrate up to the cortex or for a progenitor to proceed to the next round of division. Exposure of slices to 40%, instead of 20%, O2 significantly improved their overall thickening, cell production, and layer formation and also provided better spatial resolution by preventing the loss of transparency that accompanies cell death. © 2002 Wiley‐Liss, Inc.
Growth factor treatment promotes mobilization of young but not aged adult subventricular zone precursors in response to demyelination
Authors: Decker Laurence , Picard‐Riera Nathalie , Lachapelle François , Baron‐Van Evercooren Anne
Published: October 2002

Precursor cells of the adult mouse subventricular zone (SVZ) are mobilized and recruited by a lysolecithin (LPC)‐induced demyelination of the corpus callosum. Because age decreases the proliferation of the SVZ neural precursors as well as the potential for myelin repair of the adult central nervous system, we have compared the ability of young and aged adult neural precursors to respond to LPC‐induced demyelination. With age, the SVZ cells lost their capacity to proliferate and to be recruited by the lesion. Whereas a single injection of fibroblast growth factor‐2 or transforming growth factor‐α stimulated the proliferation of SVZ and rostral migratory stream precursors in both groups of animals after demyelination, they favored recruitment at the lesion in young mice but not in aged ones. In vitro experiments using neurospheres derived from young and aged animals indicated that both populations have the same migratory performances. Our in vivo data thus suggest that aged neural precursors may loose their intrinsic capacities to respond to demyelination‐induced signals. Alternatively, their function may be altered by modification of the aged extracellular environment. © 2002 Wiley‐Liss, Inc.
The determination of beginning‐ and end‐of‐period live weights of growing pigs
Authors: Schofield C Patrick , Whittemore Colin T , Green Darren M , Pascual Mariam D
Published: October 2002

Optimisation of meat production systems depends upon accurate performance assessment. Weigh platforms in electronic feeding stations allow continuous monitoring of individual pig weight change. Such a system was compared with a conventional mechanical scale with the general result: feeding station weight (kg) = 1.02 (0.010) scale weight − 1.65 (0.766). As compared with the conventional scale measurement at start and at finish of test, back extrapolation of daily weights collected over time in electronic feeding stations may give an improved estimate of start weight, and forward extrapolation an improved estimate of final weight. Regression predictions suggest this to be the case for final weight, but for start weight both systems are open to error due to variation in weight in the early days of test. However, such variation (and its diagnostic utility) is only observable with a system that continuously monitors weight.

© 2002 Society of Chemical Industry
Diffusion of lactic acid in a buffered gel system supporting growth of Lactobacillus curvatus
Authors: Malakar Pradeep K , Zwietering Marcel H , Boom Remko M , Brocklehurst Tim F , Wilson Peter D G , Mackie Alan R , van 't Riet Klaas
Published: October 2002

Diffusion of lactic acid in a buffered gel system was investigated using fluorescent ratio imaging. The range of pH studied was from 5.5 to 3.8, which included the minimum pH of growth of Lactobacillus curvatus (4.26). Diffusion of lactic acid in this pH range was found to be Fickian and the estimated effective diffusion coefficient of lactic acid at 20 °C was 2.81 × 10−10 m2 s−1 with a standard deviation of 0.21 × 10−10 m2 s−1. This effective diffusion coefficient was estimated by fitting the one‐dimensional solution of the Fickian diffusion equation for infinite systems to a transformed experimental data set. The original data set as recorded by the fluorescent microscope consisted of concentration–distance curves at specific time intervals. This was transformed by realigning the distance scale of all the concentration–distance curves.

© 2002 Society of Chemical Industry
Acute remote ischemic preconditioning on a rat cremasteric muscle flap model
Authors: Küntscher Markus V. , Kastell Thomas , Sauerbier Michael , Nobiling Rainer , Gebhard Martha Maria , Germann Günter
Published: October 2002

A previous study showed, in a rat adipocutaneous flap model, that acute ischemic preconditioning (IP) can be achieved not only by preclamping of the flap pedicle, but also by a brief extremity ischemia prior to flap ischemia. The purpose of this study was to determine whether remote IP is also effective in other tissues such as muscle flaps. Twenty male Wistar rats were divided into three experimental groups. The rat cremaster flap in vivo microscopy model was used for assessment of ischemia/reperfusion injury. In the control group (CG, n = 8), a 2‐hr flap ischemia was induced after preparation of the cremaster muscle. In the “classic” IP group (cIP, n = 6), a brief flap ischemia of 10 min was induced by preclamping the pedicle, followed by 30 min of reperfusion. A 10‐min ischemia of the contralateral hindlimb was induced in the remote IP group (rIP, n = 6). The limb was then reperfused for 30 min. Flap ischemia and the further experiment were performed as in the CG. In vivo microscopy was performed after 1 hr of flap reperfusion in each animal. A significantly higher red blood cell velocity in the first‐order arterioles and capillaries, a higher capillary flow, and a decreased number of leukocytes adhering to the endothelium of the postcapillary venules were observed in both preconditioned groups by comparison to the control group (P < 0.05). The differences within the preconditioned groups were not significant for these parameters. Our data show that ischemic preconditioning and improvement of flap microcirculation can be achieved not only by preclamping of the flap pedicle, but also by induction of an ischemia/reperfusion event in a body area distant from the flap prior to elevation. These findings indicate that remote IP is a systemic phenomenon, leading to an enhancement of flap survival. Our data suggest that remote IP could be performed simultaneously with flap elevation in the clinical setting without prolongation of the operation and without invasive means. © 2002 Wiley‐Liss, Inc. MICROSURGERY 22:221–226 2002
Buckling analysis of composite laminates under end shortening by higher‐order shear deformable finite strips
Authors: Zou G. P. , Lam S. S. E.
Published: October 2002

A higher‐order shear deformable finite strip is developed and employed in the buckling analysis of laminated composite plates when subjected to uniform end shortening. This enables the transverse shear deformation to be accurately incorporated. The permitted laminate material properties are quite general, encompassing anisotropy and full coupling between in‐plane and out‐of‐plane behaviour. Results with respect to the number of plies, thickness of laminate and ratios of E11/E22 are presented for unsymmetric cross‐ply and angle‐ply lay‐ups and for laminates with arbitrary lay‐up arrangements. Copyright © 2002 John Wiley & Sons, Ltd.
Whatever happened to deconcentration? Recent initiatives in Cambodia
Authors: Turner Mark
Published: October 2002

Contemporary decentralization discourse and recommended practice focus on political decentralization. However, experience with such devolution has often failed to produce the anticipated developmental returns. This has prompted reconsideration of managerial or administrative decentralization (deconcentration) as a vehicle for improving services and encouraging popular participation. A range of potential benefits is available from deconcentration. Cambodia has embarked on a cautious policy of decentralization involving weak devolution and deconcentration. But it is from deconcentration that the government expects the greatest developmental gains. Initial results are encouraging but difficulties confront the deconcentration experiment. These include the piecemeal nature of the initiative, unfamiliar financial management practices, deeply embedded patterns of hierarchy in society and state, and limited managerial capacity. Nevertheless, it appears that incremental deconcentration may offer a useful policy alternative for countries such as Cambodia which are engaged in slowly building the infrastructure of a modern state following debilitating internal conflict. Copyright © 2002 John Wiley & Sons, Ltd.
Genomic gain of PIK3CA and increased expression of p110alpha are associated with progression of dysplasia into invasive squamous cell carcinoma
Authors: Woenckhaus Joachim , Steger Klaus , Werner Eva , Fenic Irina , Gamerdinger Ulrike , Dreyer Thomas , Stahl Ulrich
Published: October 2002

PIK3CA, encoding the catalytic subunit p110α of phosphatidylinositol 3‐kinase (PI3K), is activated in malignant diseases. However, the role of the PIK3CA gene aberrations for tumourigenesis of head and neck squamous cell carcinoma (HNSCC) is to date unclear. The present study was designed to determine the genomic aberration of PIK3CA in invasive HNSCC and dysplastic precursor lesions by fluorescence in situ hybridization (FISH) with a YAC probe, containing the PIK3CA gene, on isolated interphase nuclei from histomorphologically well‐defined regions of formalin‐fixed tissue sections and to compare these data with protein and mRNA expression of p110α. The mRNA and protein levels of p110α were assessed, respectively, by in situ hybridization and immunohistochemistry on consecutive tissue sections. Copy number gains at 3q26 were observed in one of six low‐to‐moderate dysplasias (17%) and in seven of nine high‐grade dysplasias (78%), as well as in 11 carcinomas (100%). In addition, one of seven high‐grade dysplasias (14%) and 6 of 11 carcinomas (55%) had amplifications of 3q26. The majority of cases with copy number gain in more than 50% of the cells and/or amplification in more than 10% of cells showed increased p110α mRNA and protein expression, whereas only two cases (18%) (one high‐grade dysplasia and one carcinoma) with no gain or low‐level gain displayed increased p110α protein expression. These data suggest that 3q26 copy number gain and amplification represent early genomic aberrations in HNSCC carcinogenesis. In addition, p110α mRNA and protein expression in HNSCC may be regulated by these genomic aberrations as well as by epigenetic events. Copyright © 2002 John Wiley & Sons, Ltd.
Transforming growth factor‐β receptors in self‐limited vs. chronic progressive nephritis in rats
Authors: Watanabe Takuya , Yamamoto Tatsuo , Ikegaya Naoki , Fujigaki Yoshihide , Suzuki Hiroyuki , Togawa Akashi , Fukasawa Hirotaka , Nagase Mitsumasa , Hishida Akira
Published: October 2002

Increases in transforming growth factor‐β (TGF‐β) expression and extracellular matrix accumulation are transient in acute self‐limited mesangial proliferative glomerulonephritis induced by a single injection of anti‐thymocyte serum (ATS), while these increases persist following repeated injections that produce chronic progressive sclerosing glomerulonephritis with tubulointerstitial lesions. However, little is known about the expression of TGF‐β receptors (TβRs) in cells involved in the proliferative and sclerosing renal lesions. A study of protein and mRNA expression for type I (TβRI), type II (TβRII), and type III (TβRIII) TβR in both forms of nephritis was therefore carried out by immunohistochemistry and in situ hybridization. Inhibition of cell proliferation and stimulation of matrix production by TGF‐β1 were assessed in isolated glomeruli using [3H]thymidine incorporation and [3H]proline metabolic labelling, respectively. In acute self‐limited nephritis, expression of TβRI, TβRII, and TβRIII increased in the glomerular and Bowman's capsular epithelial cells comprising the glomerular tuft adhesions to Bowman's capsules. However, TβRII expression was not prominent in proliferating mesangial cells. Glomeruli isolated from rats with acute self‐limited nephritis at day 7, when mesangial cell proliferation was maximal, were partially resistant to the mitoinhibitory effects of TGF‐β1. In contrast, expression of all three TβRs was elevated in glomerular and tubulointerstitial lesions in chronic progressive nephritis, and glomeruli isolated from rats with chronic progressive nephritis 7 days after the second ATS injection were sensitive to TGF‐β1. These data suggest that distinct cellular responses to TGF‐β1 resulting from differential expression of TβR underlie the difference between acute self‐limited mesangial proliferative and chronic progressive sclerosing ATS nephritis in the development of proliferative and sclerotic renal lesions. Copyright © 2002 John Wiley & Sons, Ltd.
Regulation of IGFBP‐5 expression during tumourigenesis and differentiation of oral keratinocytes
Authors: Lin Shu‐Chun , Wang Chiung‐Pei , Chen Yi‐Man , Lu Suu‐Yi , Fann Ming‐Ji , Liu Chung‐Ji , Kao Shou‐Yen , Chang Kuo‐Wei
Published: October 2002

To identify molecular events involved in the pathogenesis of oral squamous cell carcinoma (OSCC), genes differentially expressed in OSCC and non‐cancerous matched tissue (NCMT) samples were analysed using a subtractive hybridization strategy. NCMT‐enriching clones that have been linked to suppressor pathway in previous studies were subjected to advanced analyses. Complete absence of insulin‐like growth factor binding protein‐5 (IGFBP‐5) expression at both the mRNA and the protein level was identified in nearly all (5/6) OSCC cell lines with the exception of the SCC25 cell line, which exhibited high IGFBP‐5 expression. However, this protein is consistently present in cultured normal human oral keratinocytes (NHOKs). Immunohistochemistry revealed moderate to strong cytoplasmic immunoreactivity of IGFBP‐5 in the stratum spinosum and stratum granulosum in the vast majority of NCMT samples. A remarkable reduction in IGFBP‐5 immunoreactivity was detected in 56% (26/46) of OSCC samples, compared with the corresponding NCMT (p < 0.0001). Induction of differentiation in both NHOKs and SCC25 up‐regulated IGFBP‐5 expression. Administration of a green tea compound with anti‐cancer properties, (–)‐epigallocatechin 3‐gallate, at a concentration of 5–20 µg/ml also up‐regulated IGFBP‐5 expression in NHOKs in a dose‐dependent manner. The findings suggest that IGFBP‐5 may be an important factor in the differentiation of oral keratinocytes and that down‐regulation of IGFBP‐5 may be involved in the neoplastic transformation of oral keratinocytes. Copyright © 2002 John Wiley & Sons, Ltd.
Model misspecification sensitivity analysis in estimating causal effects of interventions with non‐compliance
Authors: Jo Booil
Published: October 2002

Randomized trials often face complications in assessing the effect of treatment because of study participants' non‐compliance. If compliance type is observed in both the treatment and control conditions, the causal effect of treatment can be estimated for a targeted subpopulation of interest based on compliance type. However, in practice, compliance type is not observed completely. Given this missing compliance information, the complier average causal effect (CACE) estimation approach provides a way to estimate differential effects of treatments by imposing the exclusion restriction for non‐compliers. Under the exclusion restriction, the CACE approach estimates the effect of treatment assignment for compliers, but disallows the effect of treatment assignment for non‐compliers. The exclusion restriction plays a key role in separating outcome distributions based on compliance type. However, the CACE estimate can be substantially biased if the assumption is violated. This study examines the bias mechanism in the estimation of CACE when the assumption of the exclusion restriction is violated. How covariate information affects the sensitivity of the CACE estimate to violation of the exclusion restriction assumption is also examined. Copyright © 2002 John Wiley & Sons, Ltd.
Structure of the nucleotide‐binding domain in the β‐subunit of Escherichia coli F1‐ATPase
Authors: Duncan Thomas M. , Parsonage Derek , Senior Alan E.
Published: October 2002

We propose a working model for the tertiary structure of the nucleotide‐binding domain of the β‐subunit of E. coli F1‐ATPase, derived from secondary structure prediction and from comparison of the amino acid sequence with the sequences of other nucleotide‐binding proteins of known three‐dimensional structure. The model is consistent with previously published results of specific chemical modification studies and of analyses of mutations in the β‐subunit and its implications for subunit interactions and catalytic mechanism in F1‐ATPases are discussed.
Gold sodium thiomalate activates latent human leukocyte collagenase
Authors: Lindy Seppo , Sorsa Timo , Suomalainen Kimmo , Turto Heikki
Published: October 2002

Gold sodium thiomalate, a drug used widely in the therapy of rheumatoid arthritis, was found to be an activator of latent human polymorphonuclear leukocyte collagenase. The activation was demonstrated by two distinct and independent collagenase assays: (i) by recording with a spectrophotometer at 227 nm the enzyme‐induced increase in ultraviolet difference absorbance of native type I collagen connected to the cleavage of collagen at 37°C[(1986) Eur. J. Biochem. 156, 1‐4] and (ii) by SDS‐polyacrylamide gel electrophoresis analysis of formation of specific products of collagen resulting from collagenase cleavage at 25°C. Activation of latent collagenase by gold sodium thiomalate appeared to be of the same magnitude as by the known activator phenylmercuric chloride.
cDNA sequence of human β‐preprotachykinin, the common precursor to substance P and neurokinin A
Authors: Harmar A.J. , Armstrong A. , Pascall J.C. , Chapman K. , Rosie R. , Curtis A. , Going J. , Edwards C.R.W. , Fink G.
Published: October 2002

The nucleotide sequence of cDNA encoding the human substance P precursor, β‐preprotachykinin (β‐PPT), has been determined. The source of mRNA was a human laryngeal carcinoid tumour that contained a high concentration of immunoreactive substance P. The human β‐PPT polypeptide is 129 amino acids long and contains regions encoding substance P and neurokinin A, each flanked by basic amino acid residues. Residues 72–107 of the human β‐PPT polypeptide encode the sequence of neuropeptide K, an N‐terminally extended form of neurokinin A recently isolated from porcine brain.
Retinal photoreceptor neurons and pinealocytes accumulate mRNA for interphotoreceptor retinoid‐binding protein (IRBP)
Authors: van Veen Theo , Katial Albine , Shinohara Toshimichi , Barrett D.J. , Wiggert B. , Chader Gerald J. , Nickerson John M.
Published: October 2002

We have utilized cDNA probes and in situ hybridization techniques to define the subcellular localization of interphotoreceptor retinoid‐binding protein (IRBP) mRNA in bovine and monkey retinas. Results suggest that the mRNA is mainly localized in rod photoreceptor neurons within the outer nuclear layer of the retina. IRBP mRNA is also abundant in cells of the pineal gland, strengthening the analogy between rod photoreceptor cells and pinealocytes.
all‐trans‐Retinoic acid inhibits the appearance of two phorbol ester‐induced ornithine decarboxylase mRNAs in mouse epidermis
Authors: Kumar S. , de Vellis J. , Lowe N.J. , Weingarten D.P.
Published: October 2002

Two ornithine decarboxylase mRNA species are seen in mouse epidermis in response to the topical application of the phorbol ester tumor promoter, 12‐O‐tetradecanoyl phorbol‐13‐acetate (TPA). This induction occurs in a time‐ and dose‐dependent fashion and appears to be relatively specific as α‐actin mRNA reveals no change with treatment. Both mRNA species are inhibited in a dose‐dependent manner by treating mouse skin with all‐trans‐retinoic acid prior to TPA. These results indicate that these two compounds are active at the transcriptional level.
Corticotropin‐releasing factor mRNA in the hypothalamus is affected differently by drinking saline and by dehydration
Authors: Scott Young W.
Published: October 2002

Corticotropin‐releasing factor (CRF) stimulates the synthesis and release of adrenocorticotropin in the anterior pituitary and may help maintain fluid and electrolyte balance. ‘Salt‐loaded’ rats had an increase in CRF mRNA in hypothalamic magnocellular neurons of the paraventricular and supraoptic nuclei and a decrease in message in the parvocellular paraventricular neurons. After salt‐loaded rats were adrenalectomized, CRF mRNA increased in the parvocellular cells. In contrast to salt loading, water deprivation lead to a decrease in CRF mRNA in magnocellular and parvocellular neurons. These results show that CRF synthesis within separate populations of hypothalamic neurons is regulated differently under various conditions.
Structural properties of connectin studied by ultraviolet resonance Raman spectroscopy and infrared dichroism
Authors: Uchida Kiyoshi , Harada Issei , Nakauchi Yuni , Maruyama Koscak
Published: October 2002

Ultraviolet resonance Raman spectra of solubilized connectin indicated the presence of β‐sheets and hydrogen‐bonded irregular structures. Some Trp and Tyr sidechains are located in hydrophobic environments and some NHs of mainchain amides and Trp indoles are not easily reached by solvent water, suggesting the presence of folded structures constructed of the β‐ and irregular parts. Infrared spectra showed an abundance of β‐sheets in a connectin fiber, some of which were aligned with their mainchain axes parallel to the fiber axis. Thus, the β‐spiral structure proposed for elastin is improbable in connectin. This conclusion is also supported by their different amide III frequencies in the visible Raman spectra. A possible filamentous structure of repeated domains, consisting of β‐sheets and irregular parts, is discussed.
Dynamic light scattering studies of the aggregation of lysozyme under crystallization conditions
Authors: Skouri Mohammed , Delsanti Michel , Munch Jean-Pierre , Lorber Bernard , Giegé Richard
Published: October 2002

The intensity autocorrelation functions of light scattered by lysozyme solutions under pre‐crystallization conditions in NaCl‐containing media were recorded at scattering angles from 20° to 90°. The measurements, conducted on freshly prepared protein solutions supersaturated more than 3‐fold, indicate the simultaneous presence of two scatterer populations which can be assigned to individual protein molecules and to large particles. When solutions are undersaturated, or slightly supersaturated, light scattering only reveals the presence of the small scatterers. In the supersaturated medium, where aggregates were detected, lysozyme crystals grew in a time‐span of 1–3 days after the scattering experiments. These results are correlated with the nucleation step during protein crystallization.
PGE2 regulates cholecystokinin‐octapeptide (CCK‐8)‐stimulated Cl− conductance in isolated zymogen granules from rat pancreas
Authors: Plusczyk Thorsten , Piiper Albrecht , Schulz Irene
Published: October 2002

In this study we have examined the effects of prostaglandin E2 (PGE2), the cyclooxygenase inhibitor, indomethacin, and a protein kinase A inhibitor (PKA‐I) on the Cl− conductance in isolated zymogen granules (ZG) from cholecystokinin octapeptide (CCK‐8) pre‐stimulated pancreatic acini. The Cl− conductance in isolated ZG from CCK‐8 pre‐stimulated rat pancreatic acini increases with increasing CCK‐8 concentrations and decreases at supramaximal CCK‐8 concentrations. The basal and CCK‐8‐stimulated Cl− conductance in ZG is inhibited by pretreatment of acini with PGE2 (10−6 M). This PGE2‐induced inhibition is abolished in the presence of PKA‐I (20 U/ml). Furthermore, pretreatment of acini with indomethacin (10−5 M) or PKA‐I (20 U/ml) abolishes the decrease in the Cl− conductance at supramaximal CCK‐8 concentrations (10−9 M). We conclude that the inhibition of the Cl− conductance in isolated ZG at high CCK‐8 concentrations is mediated by an enhanced production of PGE2, and that PGE2 operates by stimulating adenylate cyclase (AC) with a consequent rise in cAMP and activation of PKA.
RNA editing in ATPase subunit 6 mRNAs in Oenothera mitochondria
Authors: Schuster Wolfgang , Brennicke Axel
Published: October 2002

The open reading frame encoding ATPase subunit 6 in Oenothera mitochondria is edited at 21 positions in all cDNA clones investigated. Only one of these events is silent, all others improve similarity between the homologous polypeptides of other species. The introduction of a new UAA termination codon shortens the polypeptide by 35 amino acids to a carboxy terminus conserved in other species. In one of the cDNA clones, an additional editing event was observed resulting in a premature UAA termination codon in the amino terminal region.
Xylose‐specific antibodies as markers of subcompartmentation of terminal glycosylation in the Golgi apparatus of sycamore cells
Authors: Lainé Anne-Catherine , Gomord Véronique , Faye Loïc
Published: October 2002

Antibodies specific for xylose‐containing plant complex N‐linked glycans are used for indirect immunolocalization of xylosyltransferase in sycamore cells. The use of high pressure freezing and freeze substitution for sample preparation resulted in very good morphological preservation of the different Golgi cisternae. Xylosyltransferase shows a diffuse distribution all over the Golgi stacks and xylosylation appears to be an early processing event that is initiated in the cis Golgi compartment.
The proton pump bacteriorhodopsin is a photoreceptor for signal transduction in Halobacterium halobium
Authors: Bibikov Sergei I. , Grishanin Ruslan N. , Marwan Wolfgang , Oesterhelt Dieter , Skulachev Vladimir P.
Published: October 2002

Halobacterium halobium swims by rotating its polarly inserted flagellar bundle. The cells are attracted by green‐to‐orange light which they can use for photophosphorylation but flee damaging blue or ultraviolet light. It is generally believed that this kind of ‘colour vision’ is achieved by the combined action of two photoreceptor proteins, sensory rhodopsins‐I and ‐II, that switch in the light the rotational sense of the bundle and in consequence the swimming direction of a cell. By expressing the bacteriorhodopsin gene in a photoreceptor‐negative background we have now demonstrated the existence of a proton‐motive force sensor (protometer) and the function of bacteriorhodopsin as an additional photoreceptor covering the high intensity range. When the bacteriorhodopsin‐generated proton‐motive force drops caused by a sudden decrease in light intensity, the cells respond by reversing their swimming direction. This response does not occur when the proton‐motive force is saturated by respiration or fermentation.
The menopause revisited
Authors: Samsioe G.
Published: October 2002

The continuing growth of female life expectancy has resulted in a marked increase of women in years beyond the menopause. Women can nowadays expect to live one‐third of their lives in a potential hormonal deficiency state. Women over 50 comprise 17% of the total population of any country in the modern western world. Any decision regarding their health issues will have a great impact on our limited health care resources. There is no doubt that estrogen replacement therapy effectively mitigates hot flushes and other vasomotor symptoms and more effectively so than other treatment modalities. Vasomotor symptoms affect more than half of the female population around the menopause with a mean duration of 2–3 years. When used to treat vasomotor symptoms hormone replacement therapy has repeatedly been shown to be cost effective. It is also well documented that hormone replacement therapy effectively prevents bone loss and osteoporotic fractures as well as heart disease. The majority of cases of both fractures and heart disease occur at ages over 75 and concern has been expressed if treatment from the menopausal age to the onset of fractures or heart disease is cost effective with regard to the perceived increase in risk of side effects, especially breast cancer. One important aspect in this scenario is the control system that we impose on women on HRT. Given our present preparations women are recommended an annual check‐up. If the number of office procedures and visits to the clinic cannot be substantially reduced long‐term therapy with HRT is not cost effective. An exception from this rule is the reatment of urogenital estrogen deficiency using low dose vaginal estrogens. Systemic concentrations of estrogens following such administration are negligible. Hence, low dose estrogen topical applications can be made an OTC preparation. As no control system is needed this therapy seems to be highly cost effective. The pharmaceutical industry is urged to produce better products so that side effects such as bleeding problems leading to a number of visits to the clinic and fear of cancer among women can be avoided. Recent data also imply that estrogen treatment may confer protection against Alzheimer's disease. Breast cancer is the remaining controversy even if recent data imply that estrogens could be given to women operated on for breast cancer without increasing the risk of a relapse.
A comparison of three tests for determining fetal pulmonary maturity
Authors: Rodriguez‐Macias K.A.
Published: October 2002

Objective: The aim of the study was to determine the sensitivity, specificity and predictive value of two simple and cheap diagnostic tests of fetal pulmonary maturity (FPM) and compare them with the more complex and expensive phospholipid profile (PP), which includes the determination of phosphatidyl glycerol (PG) and the lecithin/sphingomyelin (L/S) ratio. Methods: Samples of amniotic fluid were obtained from 70 patients between the 26th and 38th weeks of pregnancy. The PP, shake test and tap test at 2, 5 and 10 min were determined. All patients gave birth within 12 h following these tests and none received corticoids during this period. The neonates were observed for 72 h. The results of the tests were compared with the incidence of neonatal respiratory distress syndrome. Results: The sensitivity of the shake test was 92% for FPM, as was the sensitivity of PP, while for the tap test at 2, 5 and 10 min it was between 96 and 100%. In terms of specificity, the 5‐ and 10‐min tap test diagnoses were 100% correct for immaturity while all other tests were 60–78% correct. The predictive value for maturity was 100% for the tap test (5 and 10 min), 67% for the 2‐min tap test, 64% for PG, 60% for L/S and 56% for the shake test. Conclusion: The tap test is a good alternative to other diagnostic methods of FPM. Its use at the bedside, its rapidity and low cost enhance its value.
Calf thymus Hsc70 and Hsc40 can substitute for DnaK and DnaJ function in protein renaturation but not in bacteriophage DNA replication
Authors: Ziemienowicz Alicja , Konieczny Igor , Hübscher Ulrich
Published: October 2002

Calf thymus (ct) Hsc70 has been shown previously to reactivate heat‐inactivated prokaryotic and eukaryotic enzymes, while DnaK was able to reactivate solely prokaryotic enzymes. Here, we report on isolation from calf thymus of a DnaJ homolog, ctHsc40, and on testing of its cooperative function in three different assays: (i) reactivation of heat‐inactivated DNA polymerases, (ii) stimulation of the ATPase activity of ctHsc70 chaperone, and (iii) replication of bacteriophage λ DNA. Surprisingly, ctHsc70/ctHsc40 chaperones were found to reactivate the denatured prokaryotic and eukaryotic enzymes but not to promote bacteriophage λ DNA replication, suggesting species specificity in DNA replication.
The novel co‐activator CRABPII binds to RARα and RXRα via two nuclear receptor interacting domains and does not require the AF‐2 ‘core’
Authors: Bastie Jean-Noël , Despouy Gilles , Balitrand Nicole , Rochette-Egly Cécile , Chomienne Christine , Delva Laurent
Published: October 2002

We identify the RARα, RXRα and CRABPII domains required for the physical interaction of these proteins. On RARα and RXRα, the sequences correspond to the DEF and DE domains, respectively, but the interaction with CRABPII does not require the AF‐2AD ‘core’. On CRABPII, two interacting domains are identified (NRID1 and NRID2), one of which contains the only enhancement transactivation domain of CRABPII. The interaction is ligand‐independent and does not require the ligand‐binding domain of CRABPII. These results further stress that interaction of CRABPII with the nuclear receptors defines a novel level of transcriptional control.
Involvement of CCAAT/enhancer‐binding protein in regulation of the rat serine:pyruvate/alanine:glyoxylate aminotransferase gene expression
Authors: Sugiyama Tsuyoshi , Uchida Chiharu , Oda Toshiaki , Kitagawa Masatoshi , Hayashi Hideharu , Ichiyama Arata
Published: October 2002

In the rat liver, transcription of the serine:pyruvate/alanine:glyoxylate aminotransferase (SPT/AGT) gene occurs from two sites, +1 and +66, in exon 1, resulting in the formation of two mRNAs, one for a precursor of mitochondrial SPT/AGT and the other for peroxisomal SPT/AGT, respectively. In this study, we attempted to characterize the downstream promoter responsible for generation of peroxisomal SPT/AGT. The minimal downstream promoter was confined to the +21–+90 region. We demonstrated that C/EBPα and C/EBPβ bound around the downstream start site (+66) contribute to the promoter activity. The downstream promoter activity is also regulated positively by a short inverted repeat, located 20–30 bp upstream of the downstream start site, through a protein factor(s) bound to this region. On the other hand, the sequence just downstream of the start site may negatively regulate the promoter activity.
Insulators prevent transcriptional interference between two promoters in a double gene construct for transgenesis
Authors: Hasegawa Kouichi , Nakatsuji Norio
Published: October 2002

In transgenesis, the expression of two transgenes is often subject to mutual interference by each of the two expression cassettes when they are driven by different transcriptional regulatory elements in a single construct. To study this problem, we constructed vectors consisting of two expression units, one contains a strong ubiquitous promoter and the other contains a tissue‐specific transcriptional element. The expression pattern of each transgene was examined in transfected cell lines and also in transgenic mice. In both cases, two expression units in a single construct were expressed in an independent manner and were controlled by their respective regulatory element only if we placed insulators at both ends of one expression unit. These results indicate that usage of insulators is a valuable tool for transfection of double gene constructs in transgenesis.
The effect of an agglutogen on virus infection: biotinylated filamentous phages and avidin as a model
Authors: Nakamura Michihiro , Tsumoto Kouhei , Ishimura Kazunori , Kumagai Izumi
Published: October 2002

To address the effect of an agglutogen on virus infection, we studied the avidin‐associated inhibition of infection by biotinylated M13 phages (BIO‐phages). Microscopic observation of mixtures of BIO‐phages and avidin–fluorescein conjugates revealed many aggregates. Even at low phage concentrations, avidin induced inhibition of infection significantly. Anti‐M13 phage antibody also made aggregates and inhibited the infection but in a different manner from avidin. The inhibition by avidin was at ≥2 μg/ml, time dependent and marked until 10 min after the mixing of the BIO‐phages and Escherichia coli. On the other hand, antibody inhibited the infection at ≥0.1 μg/ml dose dependently, and the inhibition was time dependent and marked until 45 min after the mixing at moderate and low phage concentrations. These results indicate that avidin against BIO‐phages and antibodies are agglutogens, and the inhibition of the BIO‐phages by avidin is closely related to the tetramerization of avidin. Agglutogens may be novel alternative antiviral drugs.
Identification of a basic surface area of the FADD death effector domain critical for apoptotic signaling
Authors: Kaufmann Markus , Bozic Damir , Briand Christophe , Bodmer Jean-Luc , Zerbe Oliver , Kohl Andreas , Tschopp Jürg , Grütter Markus G.
Published: October 2002

Death effector domains (DEDs) are protein–protein interaction domains found in the death inducing signaling complex (DISC). Performing a structure‐based alignment of all DED sequences we identified a region of high diversity in α‐helix 3 and propose a classification of DEDs into class I DEDs typically containing a stretch of basic residues in the α‐helix 3 region whereas DEDs of class II do not. Functional assays using mutants of Fas‐associated death domain revealed that this basic region influences binding and recruitment of caspase‐8 and cellular FLICE inhibitor protein to the DISC.
The cytoplasmic tail peptide sequence of membrane type‐1 matrix metalloproteinase (MT1‐MMP) directly binds to gC1qR, a compartment‐specific chaperone‐like regulatory protein
Authors: Rozanov Dmitry V , Ghebrehiwet Berhane , Ratnikov Boris I , Monosov Edward Z , Deryugina Elena I , Strongin Alex Y
Published: October 2002

Membrane type‐1 matrix metalloproteinase (MT1‐MMP), a key enzyme in cell locomotion, is known to be primarily recruited to the leading edge of migrating cells. This raises a possibility that the C‐terminal cytoplasmic tail of MT1‐MMP interacts with intracellular regulatory proteins, which modulate translocations of the protease across the cell. Here, we demonstrated that MT1‐MMP via its cytoplasmic tail directly associates with a chaperone‐like compartment‐specific regulator gC1qR. Although a direct functional link between these two proteins remains uncertain, our observations suggest that the transient associations of gC1qR with the cytoplasmic tail of MT1‐MMP are likely to be involved in the mechanisms regulating presentation of the protease at the tumor cell surface.
Molecular evolution from abiotic scratch
Authors: Trifonov Edward N , Berezovsky Igor N
Published: October 2002

Recent papers on the emerging new theory of protein evolution are reviewed. Reconstruction of codon chronology, analysis of loop fold structure of proteins, and quantitative correspondence between optimal DNA ring closure size and protein domain size allow to outline specific stages in early protein evolution, each with its own size range.
The temperature dependence of the hydrogen exchange in the SH3 domain of α‐spectrin
Authors: Sadqi M. , Casares S. , López-Mayorga O. , Conejero-Lara F.
Published: October 2002

The amide hydrogen–deuterium exchange (HX) in the Src homology region 3 (SH3) domain of α‐spectrin has been measured by nuclear magnetic resonance as a function of temperature between 8 and 46°C. The analysis of the temperature dependence of HX from a statistical thermodynamic point of view has allowed us to estimate the enthalpies and entropies of the conformational processes leading to HX. The results indicate that under native conditions the domain undergoes a wide variety of conformational fluctuations, ranging from local motions, mainly located in loops, turns and chain ends and involving only low enthalpy and entropy, to extensive structural disruptions affecting its core and involving enthalpies and entropies that come fairly close to those observed during global unfolding.
End‐joining of reconstituted histone H2AX‐containing chromatin in vitro by soluble nuclear proteins from human cells
Authors: Siino Joseph S , Nazarov Igor B , Zalenskaya Irina A , Yau Peter M , Bradbury E.Morton , Tomilin Nikolai V
Published: October 2002

Non‐homologous end‐joining is an important pathway for the repair of DNA double‐strand breaks. This type of DNA break is followed by the rapid phosphorylation of Ser‐139 in the histone variant H2AX to form γ‐H2AX. Here we report efficient in vitro end‐joining of reconstituted chromatin containing nucleosomes made with either H2A or H2AX. This reaction is catalyzed by nuclear extracts from human cells and this end‐joining is not suppressed by the PI‐3 kinase inhibitor wortmannin. During the end‐joining reaction H2AX is phosphorylated at Ser‐139 as detected by immunoblot with specific antibodies and this phosphorylation is inhibited by wortmannin. Therefore, in vitro the DNA end‐joining reaction appears to be independent of H2AX phosphorylation.
Structure and function of a protein folding machine: the eukaryotic cytosolic chaperonin CCT
Authors: Valpuesta José M. , Martı́n-Benito Jaime , Gómez-Puertas Paulino , Carrascosa José L. , Willison Keith R.
Published: October 2002

Chaperonins are large oligomers made up of two superimposed rings, each enclosing a cavity used for the folding of other proteins. Among the chaperonins, the eukaryotic cytosolic chaperonin CCT is the most complex, not only with regard to its subunit composition but also with respect to its function, still not well understood. Unlike the more well studied eubacterial chaperonin GroEL, which binds any protein that presents stretches of hydrophobic residues, CCT recognises in its substrates specific binding determinants and interacts with them through particular combinations of CCT subunits. Folding then occurs after the conformational changes induced in the chaperonin upon nucleotide binding have occurred, through a mechanism that, although still poorly defined, clearly differs from the one established for GroEL. Although CCT seems to be mainly involved in the folding of actin and tubulin, other substrates involved in various cellular roles are beginning to be characterised, including many WD40‐repeat, 7‐blade propeller proteins.
Tuning of the redox potential of the primary electron donor in reaction centres of purple bacteria: effects of amino acid polarity and position
Authors: Spiedel Diane , Jones Michael R , Robert Bruno
Published: October 2002

Mutation of residues His L168 and Phe M197 in the reaction centre from Rhodobacter sphaeroides has an unusually strong effect on the mid‐point redox potential (E
m) of the pair of bacteriochlorophylls that form the primary donor of electrons, tuning E
m over a range of nearly 250 mV. This effect is correlated to the accompanying change in the permanent dipole of the L168 or M197 residue, suggesting it is mediated by changes in charge–dipole interactions. Comparisons with mutations made at a variety of other positions show that this correlation is particular to this residue pair, perhaps reflecting their proximity to the ring I regions of the dimer bacteriochlorophylls that form the overlap region between these molecules.
A selective effect of Mpl ligand on mRNA stabilization during megakaryocyte differentiation
Authors: Kaluzhny Yulia , Hechler Beatrice , Lu Jun , Nguyen Hao G , Cataldo Leah M , Ravid Katya
Published: October 2002

Megakaryocytes, the platelet precursors, are induced to differentiate in response to Mpl ligand. Here we report that stability of the megakaryocyte‐specific platelet factor 4 (PF4) mRNA is substantially augmented in the presence of Mpl ligand. This stabilization requires protein synthesis, but the 3′‐untranslated region of PF4 mRNA is not sufficient for granting the effect. This cytokine also significantly or mildly stabilizes Mpl receptor or GAPDH mRNAs, respectively, in contrast to a previously reported lack of effect on P2Y1 receptor mRNA. Our study is the first to suggest that Mpl ligand‐induced lineage specification is also determined by message stabilization.
Endothelin suppresses cell migration via the JNK signaling pathway in a manner dependent upon Src kinase, Rac1, and Cdc42
Authors: Yamauchi Junji , Miyamoto Yuki , Kokubu Hiroshi , Nishii Hiroko , Okamoto Miyuki , Sugawara Yo , Hirasawa Akira , Tsujimoto Gozoh , Itoh Hiroshi
Published: October 2002

Cell migration is a complex phenomenon that is stimulated by chemoattractive factors such as chemokines, a family of ligands for G protein‐coupled receptors (GPCRs). In contrast, factors that suppress cell migration, and the mechanism of their action, remain largely unknown. In this study, we show that endothelin, a GPCR ligand, inhibits cell motility in a manner dependent upon signaling through the c‐Jun N‐terminal kinase (JNK) pathway. We further demonstrate that this effect is dependent upon Src kinase and small GTPases Rac1 and Cdc42. These findings provide new insight into GPCR‐mediated regulation of cell migration.
Inhibition of human immunodeficiency virus type 1 replication by P‐stereodefined oligo(nucleoside phosphorothioate)s in a long‐term infection model
Authors: Inagawa Takubumi , Nakashima Hideki , Karwowski Boleslaw , Guga Piotr , Stec Wojciech J. , Takeuchi Hiroaki , Takaku Hiroshi
Published: October 2002

Oligo(nucleoside phosphorothioate)s (S‐ODNs), if prepared by conventional methods, consist of a mixture of diastereomers by virtue of the asymmetry of the phosphorus atom involved in the internucleotide linkages. This may affect the stability of the complexes formed between S‐ODNs and complementary oligoribonucleotides, which is commonly accepted as the most important factor in determining the efficacy of an antisense approach. Using HIV‐1‐infected MOLT‐4 cells via a long‐term culture approach, we studied the influence of the P‐chirality sense of stereodefined 28mer oligo(nucleoside phosphorothioate)s, [All‐Rp]‐S‐ODN‐gag‐28‐AUG and [All‐Sp]‐S‐ODN‐gag‐28‐AUG, complementary to the sequence starting at the AUG initiation codon of the gag mRNA of HIV‐1, upon the anti‐HIV‐1 activity. The [All‐Sp]‐S‐ODN‐gag‐28‐AUG at a low concentration of 0.5 μM can completely suppress HIV‐1gag p24 antigen expression in HIV‐1‐infected MOLT‐4 clone 8 cells for 32 days. Cells treated with [All‐Rp]‐S‐ODN‐gag‐28‐AUG (0.5 μM) showed a high level of the antigen expression at day 16. Furthermore, satisfactory suppression could not be achieved from a random [Mix]‐S‐ODN‐gag‐28‐AUG, consisting of a diastereomeric mixture of the oligonucleotides. Our results suggest that chemotherapy based upon the use of stereodefined antisense [All‐Sp] S‐ODN may be a more effective method for reducing the viral burden in HIV‐1‐infected individuals.
Glycine 30 in iberiotoxin is a critical determinant of its specificity for maxi‐K versus KV channels
Authors: Schroeder Nathan , Mullmann Theodore J , Schmalhofer W.A , Gao Ying-Duo , Garcia Maria L , Giangiacomo Kathleen M
Published: October 2002

Iberiotoxin (IbTX) is a remarkably selective α‐K toxin peptide (α‐KTx) inhibitor of the maxi‐K channel. In contrast, the highly homologous charybdotoxin inhibits both the maxi‐K and KV1.3 channels with similar high affinity. The present study investigates the molecular basis for this specificity through mutagenesis of IbTX. The interactions of mutated peptides with maxi‐K and KV1.3 channels were monitored through dose‐dependent displacement of specifically bound iodinated α‐KTx peptides from membranes expressing these channels. Results of these studies suggest that the presence of a glycine at position 30 in IbTX is a major determinant of its specificity while the presence of four unique acidic residues in IbTX is not.