bluuebunny/crossref_metadata_2025 huggingface dataset:
Notch signaling promotes astrogliogenesis via direct CSL‐mediated glial gene activation
Authors: Ge Weihong , Martinowich Keri , Wu Xiangbing , He Fei , Miyamoto Alison , Fan Guoping , Weinmaster Gerry , Sun Yi Eve
Published: October 2002

In the developing central nervous system (CNS), Notch signaling preserves progenitor pools and inhibits neurogenesis and oligodendroglial differentiation. It has recently been postulated that Notch instructively drives astrocyte differentiation. Whether the role of Notch signaling in promoting astroglial differentiation is permissive or instructive has been debated. We report here that the astrogliogenic role of Notch is in part mediated by direct binding of the Notch intracellular domain to the CSL DNA binding protein, forming a transcriptional activation complex onto the astrocyte marker gene, glial fibrillary acidic protein (GFAP). In addition, we found that, in CSL–/– neural stem cell cultures, astrocyte differentiation was delayed but continued at a normal rate once initiated, suggesting that CSL is involved in regulating the onset of astrogliogenesis. Importantly, although the classical CSL‐dependent Notch signaling pathway is intact and able to activate the Notch canonical target promoter during the neurogenic phase, it is unable to activate the GFAP promoter during neurogenesis. Therefore, the effect of Notch signaling on target genes is influenced by cellular context in regulation of neurogenesis and gliogenesis. © 2002 Wiley‐Liss, Inc.
Neuronal differentiation of stem cells isolated from adult muscle
Authors: Romero‐Ramos Marina , Vourc'h Patrick , Young Henry E. , Lucas Paul A. , Wu Young , Chivatakarn Onanong , Zaman Rumina , Dunkelman Noushin , El‐Kalay Mohammad A. , Chesselet Marie‐Françoise
Published: October 2002

Lineage uncommitted pluripotent stem cells reside in the connective tissue of skeletal muscle. The present study was carried out with pluripotent stem cells (PPSCs) isolated from 6‐month old rat muscle. Before differentiation, these cells were vimentin+, CD90+, CD45−, and varied in their expression of CD34. The PPSCs were expanded as non‐adherent aggregates under similar conditions to those used to generate neurospheres from embryonic or neural stem cells. The PPSC‐derived neurospheres were positive for nestin, an early marker present in neuronal precursors, and expressed the two alternative mRNA forms of the neuroectodermal marker Pax‐6, as well as mRNA for Oct‐4, a gene related to the pluripotentiality of stem cells. To confirm their neural potential, PPSC‐derived neurospheres were plated on coated coverslips under varying conditions: Neurobasal medium with N2 or B27, and either NT3 or BDNF. After 4–6 days the cells expressed neuronal (Tuj1+, NF68), astrocytic (GFAP) and oligodendrocytic (MOSP+, MBP+) markers, both by immunocytochemistry and RT‐PCR. In addition, PPSCs were cultured as monolayers under adherent conditions, exposed to growth factors and defined differentiating conditions for 5 hr, and subsequently kept for 2 days in a maturation medium. At this point they gave rise to a mixed population of early neural progenitors (Nestin+ or NG2+), immature and mature neurons (Tuj1+ and NF145+) and myelin producing oligodendrocytes (CNPase + and MOSP+). Our study shows that PPSCs present in adult muscle can overcome germ lineage restrictions and express the molecular characteristics of brain cells. Therefore, PPSCs isolated from adult muscle could provide a novel source for autologous cell replacement in neurodegenerative and demyelinating diseases. © 2002 Wiley‐Liss, Inc.
Progenitor cells derived from the adult human subcortical white matter disperse and differentiate as oligodendrocytes within demyelinated lesions of the rat brain
Authors: Windrem Martha S. , Roy Neeta S. , Wang Jeremy , Nunes Marta , Benraiss Abdellatif , Goodman Robert , McKhann Guy M. , Goldman Steven A.
Published: October 2002

A distinct population of white matter progenitor cells (WMPCs), competent but not committed to generate oligodendrocytes, remains ubiquitous in the adult human subcortical white matter. These cells are present in both sexes and into senescence and may constitute as much as 4% of the cells of adult human capsular white matter. Transduction of adult human white matter dissociates with plasmids bearing early oligodendrocytic promoters driving fluorescent reporters permits the separation of these cells at high yield and purity, as does separation based on their expression of A2B5 immunoreactivity. Isolates of these cells survive xenograft to lysolecithin‐demyelinated brain and migrate rapidly to infiltrate these lesions, without extending into normal white matter. Within several weeks, implanted progenitors mature as oligodendrocytes, and develop myelin‐associated antigens. Lentiviral tagging with green fluorescent protein confirmed that A2B5‐sorted progenitors develop myelin basic protein expression within regions of demyelination and that they fail to migrate when implanted into normal brain. Adult human white matter progenitor cells can thus disperse widely through regions of experimental demyelination and are able to differentiate as myelinating oligodendrocytes. This being the case, they may constitute appropriate vectors for cell‐based remyelination strategies. © 2002 Wiley‐Liss, Inc.
Size distribution of retrovirally marked lineages matches prediction from population measurements of cell cycle behavior
Authors: Cai Li , Hayes Nancy L. , Takahashi Takao , Caviness Verne S. , Nowakowski Richard S.
Published: October 2002

Mechanisms that regulate neuron production in the developing mouse neocortex were examined by using a retroviral lineage marking method to determine the sizes of the lineages remaining in the proliferating population of the ventricular zone during the period of neuron production. The distribution of clade sizes obtained experimentally in four different injection–survival paradigms (E11–E13, E11–E14, E11–E15, and E12–E15) from a total of over 500 labeled lineages was compared with that obtained from three models in which the average behavior of the proliferating population [i.e., the proportion of cells remaining in the proliferative population (P) vs. that exiting the proliferative population (Q)] was quantitatively related to lineage size distribution. In model 1, different proportions of asymmetric, symmetric terminal, and symmetric nonterminal cell divisions coexisted during the entire developmental period. In model 2, the developmental period was divided into two epochs: During the first, asymmetric and symmetric nonterminal cell divisions occurred, but, during the second, asymmetric and symmetric terminal cell divisions occurred. In model 3, the shifts in P and Q are accounted for by changes in the proportions of the two types of symmetric cell divisions without the inclusion of any asymmetric cell divisions. The results obtained from the retroviral experiments were well accounted for by model 1 but not by model 2 or 3. These findings demonstrate that: 1) asymmetric and both types of symmetric cell divisions coexist during the entire period of neuronogenesis in the mouse, 2) neuron production is regulated in the proliferative population by the independent decisions of the two daughter cells to reenter S phase, and 3) neurons are produced by both asymmetric and symmetric terminal cell divisions. In addition, the findings mean that cell death and/or tangential movements of cells in the proliferative population occur at only a low rate and that there are no proliferating lineages “reserved” to make particular laminae or cell types. © 2002 Wiley‐Liss, Inc.
High apparent ileal digestibility of amino acids in raw and germinated faba bean (Vicia faba)‐ and chickpea (Cicer arietinum)‐based diets for rats
Authors: Rubio Luis A , Muzquiz Mercedes , Burbano Carmen , Cuadrado Carmen , Pedrosa Mercedes M
Published: October 2002

The effects of germinated faba bean (Vicia faba minor var Alameda)‐ and chickpea (Cicer arietinum kabuli var Athenas)‐based diets on performance, N utilisation, amino acid ileal digestibility and plasma concentrations were studied in growing male Wistar rats. Raw and germinated faba bean and chickpea seed meals were incorporated in essential amino acid‐supplemented and energy‐equalised diets as the only sources of protein. A lactalbumin‐based diet equalised in protein (100 g kg−1) and digestible energy (15.5 kJ g−1) with the test diets was used as control. Weight gains, gain/feed ratios, N retention and NPU values of animals fed raw or germinated legumes were lower than those of rats given the control (lactalbumin) diet, while faecal dry weights were higher. However, N retention and NPU values of rats fed germinated faba bean diets were higher and faecal dry weights lower than those of animals fed non‐germinated faba bean diets. Liver and thymus fresh and dry relative weights were lower than controls for all legume diets, whilst small intestine, caecum and colon relative weights were higher than controls for non‐germinated faba beans and chickpeas either germinated or not. Caecum and colon fresh and dry relative weights of rats fed germinated faba bean seeds were lower than those of rats fed non‐germinated faba beans and not different from controls. Except for aspartic acid and proline in rats fed raw faba bean diets, apparent ileal digestibilities of amino acids were not different or higher in rats fed legume diets as compared with controls. Except for arginine, apparent ileal amino acid digestibility was higher for germinated than for non‐germinated faba bean seeds. The amounts of glycine, arginine and ornithine in plasma significantly increased whilst those of threonine, alanine, leucine and lysine decreased with respect to controls in faba bean‐ or chickpea‐fed rats, either germinated or not. We conclude that (a) germination of faba bean seeds gave rise to a significant (P < 0.01) but limited improvement in performance, N retention and apparent ileal digestibilities of some amino acids in growing rats compared with non‐germinated seeds, while there was no detectable effect in the case of germinated chickpea seeds, and (b) the apparent ileal amino acid digestibility of essential amino acid‐supplemented diets based on faba bean or chickpea meals, either germinated or not, is similar or even higher than controls.

© 2002 Society of Chemical Industry
Effects of type of packaging material on physicochemical and sensory characteristics of deep‐fat‐fried banana chips
Authors: Ammawath Wanna , Che Man Yaakob B , Yusof Salmah , Rahman Russly A
Published: October 2002

A storage study of deep‐fat‐fried banana chips was carried out for 8 weeks at ambient temperature (27 °C), using four types of packaging material: laminated aluminium foil (LAF), oriented polypropylene (OPP), polypropylene (PP) and low‐density polyethylene (LDPE). The physicochemical and sensory characteristics of the stored banana chips were analysed at weeks 0, 2, 4, 6 and 8. The quality parameters determined were moisture content, water activity (aw), thiobarbituric acid‐reactive substances (TBARS), texture (breaking force), colour and sensory attributes. The moisture content, aw, TBARS and breaking force values of all samples increased during storage. The colour also changed during storage, showing higher L and lower a and b values. Samples packed in LAF had the lowest moisture content, aw, TBARS and breaking force values. The most notable sensory change that occurred during storage was a decrease in crispness. Samples packed in LAF had higher scores than the other three samples, whilst LDPE gave the lowest scores for crispness as well as product colour. There were no significant differences (P > 0.05) in rancid odour among samples packed in OPP, PP and LDPE. However, there were significant differences (P < 0.05) between samples packed in LAF and the other three samples, with LAF giving the lowest rancid odour.

© 2002 Society of Chemical Industry
Development changes of anthocyanins in Vitis vinifera grapes grown in the Douro Valley and concentration in respective wines
Authors: Mateus Nuno , Machado José M , de Freitas Víctor
Published: October 2002

Grapes of Touriga Nacional and Touriga Francesa red Vitis vinifera varieties have been sampled from two vineyard sites at different altitudes during three consecutive vintage years (1997, 1998 and 1999) in the Douro Valley. The total anthocyanidin monoglucosides (AMGs) detected by HPLC/DAD and the red colour of grape skin extracts were monitored during the last month of maturation. Microvinifications were performed with the grapes studied and the resulting Port wines were analysed. Malvidin 3‐glucoside and its acylated esters were the major AMGs irrespective of cultivar at harvest date. Vineyard altitude, together with other variables, was found to be an important factor contributing to the amount of anthocyanin compounds found in grapes of Touriga Nacional and Touriga Francesa. Overall, the climatic conditions observed at higher vineyard sites appeared to be advantageous, resulting in larger amounts of AMGs in grapes. The same outcome was obtained in the resulting wines, which showed higher levels of AMGs when made from grapes grown at higher altitude, especially for Touriga Francesa.

© 2002 Society of Chemical Industry
Digestive utilisation of protein and amino acids from raw and heated lentils by growing rats
Authors: Porres Jesús M , Urbano Gloria , Fernández‐Fígares Ignacio , Prieto Carlos , Pérez Luis , Aguilera José F
Published: October 2002

The effect of autoclave treatment on the digestive utilisation of protein and amino acids from lentils was studied in growing rats. Twenty 3‐week‐old Wistar rats (mean live weight 59 ± 4.8 g) were fed two experimental diets (n = 10 rats per diet) consisting of raw lentils (Lens culinaris M, var vulgaris, cv magda‐20) (diet RL) or lentils autoclaved at 120 °C and 1 atm for 30 min (diet AL). An additional group of 10 animals was fed a low‐protein (4%) diet and used to estimate metabolic nitrogen and amino acid excretion. Autoclaving caused a 76% reduction in the levels of trypsin inhibitor activity, but failed to improve the faecal digestive utilisation of protein or total amino acids. Lowest essential amino acid true digestibility in diets RL and AL was found for cysteine (60.2 vs 60.1%) and methionine (68.9 vs 66.6%). The protein digestibility‐corrected amino acid score was 71.2 and 66.4% and the availability of sulphur amino acid‐corrected amino acid score was 57.1 and 52.1% for diets RL and AL respectively. Sulphur amino acids were the first limiting ones. Overall, autoclave treatment did not improve protein or total amino acid digestibility from lentils, but caused a significant improvement in leucine and lysine digestibility and a significant decrease in the digestive utilisation of tyrosine and methionine.

© 2002 Society of Chemical Industry
Acute remote ischemic preconditioning II: The role of nitric oxide
Authors: Küntscher Markus V. , Kastell Thomas , Altmann Jens , Menke Henrik , Gebhard Martha Maria , Germann Günter
Published: October 2002

The purpose of this study was to determine whether nitric oxide (NO) plays a role in the mechanism of acute “classic” as well as acute remote ischemic preconditioning (IP). Thirty‐two male Wistar rats were divided into five 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. The animals of group NO (n = 6) received 500 nmol/kg of the NO‐donor spermine/nitric oxide complex (Sper/NO) intravenously 30 min prior to ischemia. The group LN + P (L‐NAME + preclamping, n = 6) received 10 mg/kg Nω‐nitro‐L‐arginine methyl ester (L‐NAME) intravenously before preclamping of the flap pedicle (10‐min cycle length, 30‐min reperfusion). L‐NAME (10 mg/kg) was administered in group LN + T (L‐NAME + tourniquet, n = 6) before ischemia of the right hindlimb was induced, using a tourniquet for 10 min after flap elevation. The limb was then reperfused for 30 min. Thereafter, flap ischemia was induced in each group as in group CG. In vivo microscopy was performed after 1 hr of flap reperfusion in each animal. Group NO demonstrated a significantly higher red blood cell velocity (RBV) in the first‐order arterioles and capillaries, a higher capillary flow, and a decreased number of leukocytes adhering to the endothelium (stickers) of the postcapillary venules by comparison to all other groups (P < 0.05). The average capillary RBV and capillary flow were still higher in the CG than in the groups receiving L‐NAME (P < 0.05). The data show that NO plays an important role in the mechanism of both acute “classic” as well as acute remote IP, since the administration of a NO‐donor previous to ischemia simulates the effect of IP, whereas the nonspecific blocking of NO synthesis by L‐NAME abolishes the protective effect of flap preconditioning. © 2002 Wiley‐Liss, Inc. MICROSURGERY 22:227‐231 2002
Superficial or deep implantation of motor nerve after denervation: An experimental study—superficial or deep implantation of motor nerve
Authors: Askar Ibrahi̇m , Sabuncuoglu Bi̇zden Tavi̇l
Published: October 2002

Neurorraphy, conventional nerve grafting technique, and artificial nerve conduits are not enough for repair in severe injuries of peripheral nerves, especially when there is separation of motor nerve from muscle tissue. In these nerve injuries, reinnervation is indicated for neurotization. The distal end of a peripheral nerve is divided into fascicles and implanted into the aneural zone of target muscle tissue. It is not known how deeply fascicles should be implanted into muscle tissue. A comparative study of superficial and deep implantation of separated motor nerve into muscle tissue is presented in the gastrocnemius muscle of rabbits. In this experimental study, 30 white New Zealand rabbits were used and divided into 3 groups of 10 rabbits each. In the first group (controls, group I), only surgical exposure of the gastrocnemius muscle and motor nerve (tibial nerve) was done without any injury to nerves. In the superficial implantation group (group II), tibial nerves were separated and divided into their own fascicles. These fascicles were implanted superficially into the lateral head of gastrocnemius muscle‐aneural zone. In the deep implantation group (group III), the tibial nerves were separated and divided into their own fascicles. These fascicles were implanted around the center of the muscle mass, into the lateral head of the gastrocnemius muscle‐aneural zone. Six months later, histopathological changes and functional recovery of the gastrocnemius muscle were investigated. Both experimental groups had less muscular weight than in the control group. It was found that functional recovery was achieved in both experimental groups, and was better in the superficial implantation group than the deep implantation group. EMG recordings revealed that polyphasic and late potentials were frequently seen in both experimental groups. Degeneration and regeneration of myofibrils were observed in both experimental groups. New motor end‐plates were formed in a scattered manner in both experimental groups. However, they were more dense in the superficial implantation group than the deep implantation group. It was concluded that superficial implantation has a more powerful contractile capacity than that of deep implantation. We believe that this might arise from the high activity of glycolytic enzymes in peripheral muscle fibers of gastrocnemius muscle, decrease in insufficient intramuscular guidance apparatus, and intramuscular microneuroma formation at the insufficient neuromuscular junction since the motor nerve had less route to muscle fibers. © 2002 Wiley‐Liss, Inc. MICROSURGERY 22:242–248 2002
Numerical simulation of the fracture process in cutting heterogeneous brittle material
Authors: Liu H. Y. , Kou S. Q. , Lindqvist P.‐A.
Published: October 2002

The process of cutting homogeneous soft material has been investigated extensively. However, there are not so many studies on cutting heterogeneous brittle material. In this paper, R‐T2D (Rock and Tool interaction), based on the rock failure process analysis model, is developed to simulate the fracture process in cutting heterogeneous brittle material. The simulated results reproduce the process involved in the fragmentation of rock or rock‐like material under mechanical tools: the build‐up of the stress field, the formation of the crushed zone, surface chipping, and the formation of the crater and subsurface cracks. Due to the inclusion of heterogeneity in the model, some new features in cutting brittle material are revealed. Firstly, macroscopic cracks sprout at the two edges of the cutter in a tensile mode. Then with the tensile cracks releasing the confining pressure, the rock in the initially high confining pressure zone is compressed into failure and the crushed zone gradually comes into being. The cracked zone near the crushed zone is always available, which makes the boundary of the crushed zone vague. Some cracks propagate to form chipping cracks and some dip into the rock to form subsurface cracks. The chipping cracks are mainly driven to propagate in a tensile mode or a mixed tensile and shear mode, following curvilinear paths, and finally intersect with the free surface to form chips. According to the simulated results, some qualitative and quantitative analyses are performed. It is found that the back rake angle of the cutter has an important effect on the cutting efficiency. Although the quantitative analysis needs more research work, it is not difficult to see the promise that the numerical method holds. It can be utilized to improve our understanding of tool–rock interaction and rock failure mechanisms under the action of mechanical tools, which, in turn, will be useful in assisting the design of fragmentation equipment and fragmentation operations. Copyright © 2002 John Wiley & Sons, Ltd.
MAP kinase activation and apoptosis in lung tissues from patients with idiopathic pulmonary fibrosis
Authors: Yoshida Koichiro , Kuwano Kazuyoshi , Hagimoto Naoki , Watanabe Kentaro , Matsuba Tokuji , Fujita Masaki , Inoshima Ichiro , Hara Nobuyuki
Published: October 2002

Three major MAP kinases (MAPKs), including extracellular signal‐regulated kinase (ERK), c‐jun N‐terminal kinase (JNK), and p38 kinase (p38 MAPK), are involved in the regulation of lung inflammation and injury. This study investigated whether MAPKs are activated and associated with lung injury in lung tissues from patients with idiopathic pulmonary fibrosis (IPF). The expression of the active ERK, JNK, and p38 MAPK was examined using western blot analysis and immunohistochemistry and apoptosis was also examined by the TUNEL method, in lung tissues from ten patients with IPF obtained by thoracoscopic biopsy and in eight normal lung parenchyma specimens obtained by lobectomy for lung cancer. Activated MAPKs are significantly increased in lung homogenates from patients with IPF compared with controls. Activated ERK in epithelial and endothelial cells, but not in fibroblasts or smooth muscle cells, was decreased, accompanied by the progression of fibrosis. Activated JNK in epithelial and endothelial cells, but not in fibroblasts, was increased, accompanied by the progression of fibrosis. Activated p38 MAPK in epithelial, endothelial, smooth muscle cells, and fibroblasts was increased at the intermediate stage of fibrosis, in which the TUNEL‐positive cells were predominantly detected. This is the first study to suggest that MAPKs may be associated with the regulation of inflammation and lung injury in IPF. Copyright © 2002 John Wiley & Sons, Ltd.
Multiple symmetric lipomatosis may be the consequence of defective noradrenergic modulation of proliferation and differentiation of brown fat cells
Authors: Nisoli Enzo , Regianini Laura , Briscini Luca , Bulbarelli Alessandra , Busetto Luca , Coin Alessandra , Enzi Giuliano , Carruba Michele O
Published: October 2002

Multiple symmetric lipomatosis (MSL) is an inherited disorder in which enlarging and unencapsulated lipomas symmetrically develop in the subcutaneous tissue of the neck, shoulders, mammary, and truncal regions. In some cases, it is associated with mitochondrial DNA abnormalities. The pathogenesis of MSL is completely unknown, although the fat deposits may be due to a neoplastic‐like proliferation of functionally defective brown adipocytes. It has recently been demonstrated that the β3‐adrenergic receptor is the functionally relevant adrenergic receptor subtype in brown adipocytes and that its stimulation by noradrenaline (NA) modulates the expression of genes, such as uncoupling protein (UCP)‐1 and inducible nitric oxide synthase (iNOS), involved in fat cell proliferation and differentiation. Furthermore, Trp64Arg mutation of the β3‐adrenoceptor has been implicated in lower NA activity in adipose tissues. The aim of this study was to investigate the molecular and functional characteristics of MSL adipocytes and to analyse the effects of nitric oxide (NO) on the proliferation/differentiation of MSL adipocytes in culture, and the relevance of putative noradrenergic deficit in the development of lipomas in MSL patients. Cultured MSL adipocytes were able to synthesize UCP‐1 (the selective marker of brown adipocytes), but unlike that of normally functioning brown fat cells, the expression of the UCP‐1 gene was not significantly induced by NA. NA is also defective in inducing iNOS gene expression, thus leading to reduced NO production and a consequent reduction in the anti‐proliferative, adipogenic (mitochondrial biogenesis) effects of NA on MSL cells. Furthermore, the transcriptional peroxisome proliferator‐activated receptor γ co‐activator‐1 (PGC‐1), which plays a key role in the sympathetic‐stimulated mitochondrial biogenesis of brown adipocytes, is expressed but not induced by NA in MSL cells, as it is in brown adipocytes. The study did not find any association between β3‐adrenoceptor gene polymorphism and noradrenergic signalling defects in MSL subjects with or without mitochondrial DNA mutations. Copyright © 2002 John Wiley & Sons, Ltd.
Towards defining roles and relationships for tenascin‐C and TGFβ‐1 in the normal and neoplastic urinary bladder
Authors: Booth Catherine , Harnden Patricia , Selby Peter J , Southgate Jennifer
Published: October 2002

Tenascin‐C (TN‐C) is an extracellular matrix glycoprotein expressed along epithelial/stromal boundaries during tissue remodelling events, such as those that occur during morphogenesis, wound healing, and tumour invasion. Using clinical specimens and a range of in vitro models that simulate homeostasis, wound healing, and malignant progression, this study sought to establish the patterns of TN‐C expression in normal and neoplastic bladder and to determine the role of exogenous transforming growth factor β‐1 (TGFβ‐1), interleukin‐4 (IL‐4), basic fibroblast growth factor (bFGF), tumour necrosis factor alpha (TNFα), and interferon gamma (IFNγ) in the induction of TN‐C expression by bladder uro‐epithelial cells. The findings indicate that normal urothelial cells may express TN‐C, with both TGFβ‐1 and IL‐4 able to induce expression. TN‐C was not expressed in neoplastic urothelium, although both TN‐C and TGFβ‐1 may be involved in tissue remodelling during papillary tumour formation and invasion. Furthermore, the urothelium of high‐grade papillary tumours and carcinoma in situ specimens exhibited little TGFβ‐1 immunoreactivity, compared with the urothelium of low‐grade tumours and normal specimens, suggesting an association between TGFβ‐1 expression and urothelial differentiation. A tumour invasion model, in which established bladder cancer cell lines were seeded onto a normal bladder stroma, corroborated the evidence from the clinical specimens and demonstrated that TN‐C was strongly expressed around foci of stromal invasion. Thus, TN‐C immunoreactivity may provide an additional tool in the assessment of early stromal invasion in bladder cancer. Copyright © 2002 John Wiley & Sons, Ltd.
Numerical aberrations of chromosome 1 in cervical intraepithelial neoplasia are strongly associated with infection with high‐risk human papillomavirus types
Authors: Bulten Johan , Melchers Willem JG , Kooy‐Smits Maria M , de Wilde Peter CM , Poddighe Pino J , Robben Johanna CM , Macville Merryn VE , Massuger Leon FAG , Bakkers Judith MJE , Hanselaar Antonius GJM
Published: October 2002

The aims of this study were to assess the relationships between numerical aberrations of chromosome 1 and the presence of high‐risk human papillomavirus (HPV). Five normal samples, 11 CIN1, 13 CIN2, 18 CIN3, and nine carcinomas were studied byin situhybridization (ISH), using a DNA probe for the centromere of chromosome 1 (cen#1) and a DNA probe cocktail for HPV types 16 and 18. A short fragment polymerase chain reaction hybridization line probe assay (SPF‐PCR‐LiPA) technique was used to detect 25 HPV types. The mean number of cen#1 per nucleus (chromosome index, CI) was measured, and the fractional areas of dysplastic epithelium with HPV16/18 infection and with cen#1 aneusomy were estimated. Disomy was found in all normal epithelium and in 36% of CIN1. Tetrasomy was observed in 64% of CIN1, 15% of CIN2, and 17% of CIN3. Hyper‐tetrasomy was observed in 77% of CIN2, 83% of CIN3, and 100% of invasive carcinomas. High‐risk HPVs were present in 20%, 75%, and 94% of disomic, tetrasomic, and hyper‐tetrasomic lesions, respectively. The mean CI value was significantly higher in the lesions infected with high‐risk HPV than in the lesions not infected by high‐risk HPV (p< 0.001), due to the significantly higher prevalence of hyper‐tetrasomy. The ISH study disclosed that HPV16/18 was exclusively found within dysplastically altered epithelium. The area with aneusomy is mostly enclosed within the area infected with HPV. In 83% of the HPV16/18‐positive CIN lesions, the fractional area of HPV‐infected epithelium was equal to, or larger than, the fractional area with aneusomy. In conclusion, aneusomy for chromosome 1 is strongly associated with high‐grade CIN lesions and infection with high‐risk HPV; it is likely that the occurrence of numerical aberrations of chromosome 1 is preceded by infection with high‐risk HPV. Copyright © 2002 John Wiley & Sons, Ltd.
Radioimmunoassay for leukotriene E4
Authors: Hoppe U. , Hoppe E.M. , Peskar B.M. , Peskar B.A.
Published: October 2002

A conjugate of leukotriene (LT) E4 and bovine serum albumin (BSA) was prepared by covalently linking the free amino group of the hapten to the protein using dimethyl pimelindiimidate (DMP) as coupling reagent. Anti‐LTE4 antibodies were raised in rabbits immunized with the conjugate. Binding of[3H]LTE4 to the antibodies is inhibited by 50% with 0.63 ng LTE4, while the relative cross‐reaction of LTC4 and LTD4 is 46.3% and 12.6%, respectively. Using the radioimmunoassay release of sulfidopeptide‐LT (SP‐LT) from rat gastric mucosa incubated in vitro was determined after quantitative enzymatic conversion of SP‐LT to LTE4. It could be demonstrated that this method is suitable for determination of SP‐LT in biological material.
Sliding‐end‐labelling
Authors: Pérez-Ortin Josh E. , Estruch Francisco , Matallana Emilia , Franco Luis
Published: October 2002

A method, termed ‘sliding‐end‐labelling’, has been devised to avoid a frequent artifact in nucleosome positioning by indirect end labelling, namely the appearing of DNA fragments originated by two nuclease cuts, one of them lying within the region covered by the probe. The method is applied to the nucleosome positioning in the yeast SUC2 gene for invertase.
Early rise of cytosolic Ca2+ induced by NGF in PC12 and chromaffin cells
Authors: Pandiella-Alonso Atanasio , Malgaroli Antonio , Vicentini Lucia M. , Meldolesi Jacopo
Published: October 2002

A rise of cytosolic Ca2+ is induced by NGF in rat pheochromocytoma PC12 and bovine chromaffin cells investigated (both in suspension and while attached to polyornithine‐coated glass slides) by fluorescence techniques (with quin‐2 and fura‐2). The effect of NGF on [Ca2+]i is delayed (30–40 s of lag phase), slow (tf1/2 = 40 s), relatively small (+50‐75%) and persistent (over 10 min). It is due to Ca2+ influx (requires extra‐cellular Ca2+ > 10 μM) through a pathway different from the voltage‐gated Ca2+ channel, possibly accompanied by intracellular Ca2+ redistribution, and might play a messenger role in NGF action.
1‐O‐Alkyl‐2‐acetyl‐sn‐glyceryl‐3‐phosphorylcholine (PAF) is a minor lipid component in Tetrahymena pyriformis cells
Authors: Lekka Marilena , Tselepis Alexandros D. , Tsoukatos Demokritos
Published: October 2002

The protozoan Tetrahymena pyriformis contains 4.2 ± 2.2 ng PAF/107 cells. Only 1–3% of this lipid is released in the cell free medium. PAF production is not influenced by different extracellular Ca2+ concentrations. Cell stimulation with calcium ionophore A23187 or zymosan particles does not affect the amount of PAF either. This is the first report of a natural occurrence of PAF in a protozoan.
The ATP‐sensitivity of K+ channels in rat pancreatic B‐cells is modulated by ADP
Authors: Kakei Masafumi , Kelly Ronan P. , Ashcroft Stephen J.H. , Ashcroft Frances M.
Published: October 2002

ATP‐sensitive K+ channels in inside‐out membrane patches from dispersed rat pancreatic B‐cells were studied using patch‐clamp methods. The dose‐response curve for ATP‐induced channel inhibition was shifted to higher concentrations in the presence of ADP (2 mM). In glucose‐free solution, the total intracellular concentration of ATP was 3.8 mM and of ADP 1.5 mM; glucose (20 mM) increased ATP and decreased ADP by approx. 40%. These results suggest that both ADP and ATP may be involved in regulating the activity of the glucose‐sensitive K+ channel in intact B‐cells.
Two rules of enzyme kinetics for reversible Michaelis‐Menten mechanisms
Authors: Keleti T.
Published: October 2002

In a Michaelis‐Menten type reversible enzyme reaction (one substrate, one product) the rapid equilibrium kinetics in one direction excludes rapid equilibrium in the reverse direction. If rapid equilibrium functions in any direction, in the reverse reaction van Slyke type ‘kinetic constant’ appears in the rate equation independently of whether steady state is reached in finite time or the final equilibrium is attained at t = ∞. If the reaction proceeds in one direction with rapid equilibrium and in the reverse direction with steady‐state kinetics, the thermodynamic equilibrium of the reaction determines that a higher equilibrium concentration of product (or substrate) can be reached only with steady‐state kinetics.
Evidence for two protein‐lipoylation activities in Escherichia coli
Authors: Brookfield Dawn E. , Green Jeffrey , Ali Sohail T. , Machado Rosane S. , Guest John R.
Published: October 2002

The lipoate acyltransferase subunits of the 2‐oxo acid dehydrogenase complexes are post‐translationally modified with one or more covalently‐bound lipoyl cofactors. Two distinct lipoate‐protein ligase activities, LPL‐A and LPL‐B, have been detected in E. coli by their ability to modify purified lipoyl apo‐domains of the bacterial pyruvate dehydrogenase complex. Both enzymes require ATP and Mg2+, use L‐lipoate, 8‐methyllipoate, lipoyl adenylate and octanoyl adenylate as substrates, and both activate lipoyl‐deficient pyruvate dehydrogenase complexes. In contrast, only LPL‐B uses D‐lipoate and octanoate and there are differences in the metal‐ion and phosphate requirements. It is suggested that LPL‐B may be responsible for the octanoylation of lipoyl domains observed previously under lipoate‐deficient conditions.
Compositional properties of telomeric regions from human chromosomes
Authors: De Sario Albertina , Aïssani Brahim , Bernardi Giorgio
Published: October 2002

We have investigated the GC levels of third codon position of genes localized in G‐ (Giemsa), R‐ (reverse) and T‐ (telomeric) bands of human metaphase chromosomes, as well as the hybridization of telomeric probes on fractionated human DNA. The first set of results shows much higher GC levels for genes localized in T‐bands than in G‐ or R‐bands (the latter being higher than the former). The second set of data shows that telomeric probes corresponding to T‐bands hybridize on the GC‐richest family (H3) of isochores, whereas telomeric probes corresponding to R‐bands hybridize on GC‐rich families H1 and H2; in agreement with these findings, the telomeric repeat common to all chromosomes hybridized on isochore families H1, H2 and H3.
Nature of papain products resulting from inactivation by a peptidyl O‐acyl hydroxamate
Authors: Ménard Robert , Rong Feng  , Storer Andrew C. , Robinson Valerie J. , Smith Roger A. , Krantz Allen
Published: October 2002

Mass spectrometry has been used to provide insights into the mechanism of inhibition of cysteine proteases by a hydroxylamine derivative, CBZ‐Phe‐Gly‐NH‐O‐CO‐(2,4,6‐Me3)Ph. An oxidized form of papain resulting from the incubation of the enzyme with the peptidyl hydroxamate in the absence of a reducing agent has been identified as a sulfinic acid. The presence of a covalent enzyme‐inhibitor complex of molecular mass consistent with a sulfenamide adduct of papain could also be detected by this method. Implications on the mechanism of inactivation of cysteine proteases by peptidyl hydroxamates are discussed.
Tomato alcohol dehydrogenase
Authors: Van Der Straeten Dominique , Rodrigues Pousada Renato A. , Gielen Jan , Van Montagu Marc
Published: October 2002

We have isolated and sequenced a partial tomato alcohol dehydrogenase (Adh) cDNA clone. Expression of tomato Adh was studied at the messenger RNA level in seedlings, roots, and fruit. High induction was observed under hypoxic conditions, both in tomato seedlings and in roots. In addition, the Adh mRNA was present at the mature green and pink stage of the tomato fruit, and was highly induced in late ripening. Moreover, an artificial ripening treatment resulted in at least 50‐fold induction compared to the mature green mRNA level. Genomic DNA gel blotting suggested the presence of a multigene family for Adh in tomato.
Inositol and inositol 1,4,5‐trisphosphate content of Down syndrome fibroblasts exhibiting enhanced inositol uptake
Authors: Fruen Bradley R. , Lester Bruce R.
Published: October 2002

Fibroblasts from individuals with Down syndrome (DS; trisomy 21) exhibit increased inositol uptake. Here we examine the relationship between this increase in uptake and mass levels of free inositol and inositol 1,4,5‐trisphosphate (IP3) in DS fibroblasts. We report that human fibroblasts contain high levels of free inositol which are not significantly affected by the increase in inositol uptake associated with DS. In addition, increased uptake is accompanied by increased efflux of radiolabelled inositol from DS cells. Neither basal nor bradykinin‐stimulated IP3 levels in DS cells differ significantly from normal values. This work highlights the usefulness of the DS cell in uncovering the role of transport across the plasma membrane in cellular inositol homeostasis.
Regulatory light chain influences alterations of myosin head induced by actin
Authors: Babiychuk Eduard B. , Stepkowski Dariusz , Danilova Valentina M. , Kakol Irena
Published: October 2002

The effect of magnesium‐for‐calcium exchange and phosphorylation of regulatory light chain (LC2) on structural organization of rabbit skeletal myosin head was studied by limited tryptic digestion. In the presence of actin, exchange of magnesium bound to LC2 by calcium in dephosphorylated myosin accelerates the digestion of myosin and heavy meromysin heavy chain and increases the accumulation of a 50 kDa fragment. This effect is significantly diminished in the case of phosphorylated myosin. Thus, both phosphorylation and cation exchange influences the effect of actin binding on the structural organization of myosin head.
Possible requirement of serum progression factors for transformation of BALB/c 3T3 fibroblasts by v‐Ras p21
Authors: Harada Shun-ichi , Nishimoto Ikuo
Published: October 2002

To form colonies in soft agar, ras‐transformed 3T3 fibroblasts require serum. We examined what growth factors in serum were essential for ras‐induced transformation. Temperature‐sensitive (ts) v‐Ki‐ras‐transfected BALB/c 3T3 cells were used to strictly control both the activity of the ras protein and the cell cycle. When G
n
‐arrested ts cells were cultured with 10% serum at a permissive temperature, >50% of cells formed colonies. A similar colony‐forming activity was observed in the presence of 10% platelet‐poor plasma, but not in the presence of 10% plasma isolated from hypophysectomized rats. Inhibitors of IGF signals attenuated colony formation in the presence of serum. These data suggest that progression factors, probably IGFs, are essential components in serum for ras‐induced transformation of 3T3 fibroblasts.
Type‐1 inhibitor of plasminogen activators
Authors: Munch Mette , Christian Heegaard , Jensen Poul H. , Andreasen Peter A.
Published: October 2002

Type‐1 inhibitor of plasminogen activators (PAI‐I) occurs in purified preparations in a latent form that can be activated with denaturants; in vivo, latency is prevented by binding to vitronectin. We have compared latent, denaturant‐activated and reactive centre‐cleaved human PAI‐1 with respect to thermal stability and affinity to monoclonal antibodies. By both criteria, latent and cleaved PAI‐1 are very similar or indistinguishable, and clearly different from active PAI‐1. Our findings suggest that the conformations of latent and reactive centre‐cleaved PAI‐1 are similar and resemble the so‐called relaxed (R) serpin conformation, while that of active PAI‐1 is different and resembles the stressed S serpin conformation.
A sodium‐stimulated ATP synthase in the acetogenic bacterium Acetobacterium woodii
Authors: Heise Reno , Reidlinger Jutta , Müller Volker , Gottschalk Gerhard
Published: October 2002

Experiments with resting cells of Acetobacterium woodii were performed to elucidate the coupling ion used by the ATP synthase. A. woodii synthesized ATP in response to an artificial ΔpH, indicating the presence of a proton‐translocating ATPase. On the other hand, a ΔpNa, as well as a proton diffusion potential, could serve as a driving force for ATP synthesis with the latter strictly dependent on Na+. These results are indicative for the presence of a Na+‐translocating ATP synthase in A. woodii.
The active site structure of E. coli HPII catalase
Authors: Dawson John H. , Bracete Alma M. , Huff Ann M. , Kadkhodayan Saloumeh , Zeitler Caroline M. , Sono Masanori , Chang Chi K. , Loewen Peter C.
Published: October 2002

E. coli produces 2 catalases known as HPI and HPII. While the heme prosthetic group of the HPII catalase has been established to be a dihydroporphyrin or chlorin, the identity of the proximal ligand to the iron has not been addressed. The magnetic circular dichroism (MCD) spectrum of native ferric HPII catalase is very similar to those of a 5‐coordinate phenolate‐ligated ferric chlorin complex, a model for tyrosinate proximal ligation, as well as of chlorin‐reconstituted ferric horseradish peroxidase, a model for 5‐coordinate histidine ligation. However, further MCD comparisons of chlorin‐reconstituted myoglobin with parallel ligand‐bound adducts of the catalase clearly rule out histidine ligation in the latter, leaving tyrosinate as the best candidate for the proximal ligand.
Chloramphenicol is an inhibitor of photosynthesis
Authors: Okada Katsuhiko , Satoh Kazuhiko , Katoh Sakae
Published: October 2002

Chloramphenicol inhibited significantly but incompletely photosynthesis in leaf segments of rice. Fluorescence and polarographic experiments indicated that chloramphenicol competes with the CO2 reducing cycle for electrons from photosystem I because it serves as an electron acceptor of photosystem I and its reduction intermediate transfers its electron to molecular oxygen.
The control of cellular motility and the role of gelsolin
Authors: Grazi Enrico , Magri Ermes , Cuneo Paola , Cataldi Alberto
Published: October 2002

Solation of actin gel by gelsolin is much less efficient in the presence of a high concentration of macromolecular solutes. The rigidity of the gel formed by 12 μM actin is lowered from 4 to 0.33 dynes/cm2 by 15 nM gelsolin, while in 6% (w/v) polyethylene glycol, rigidity is lowered only from 20 to 11 dynes/cm2 by 64 nM gelsolin. Owing to the large concentration of protein, transitions in the fluid‐ and gel‐like properties of the cytoplasm are expected to be problematic when promoted by gelsolin alone.
A rapid change in phosphorylation on tyrosine accompanies fertilization of sea urchin eggs
Authors: Ciapa Brigitte , Epel David
Published: October 2002

Alterations in protein phosphorylation, particularly phosphorylation on tyrosine, frequently accompany cell change and are important agents in the cascades initiated by extracellular signals. This paper examines whether the activation of the sea urchin egg at fertilization involves an early and rapid phosphotyrosine response. Using an anti‐phosphotyrosine antibody and a rapid sampling technique, we find a very early increase in the phosphorylation on tyrosine of two proteins of approximately 91 kDa and 138 kDa. A similar phosphorylation occurs after activation of the eggs by the calcium ionophore, ionomycin, suggesting the stimulation of a Ca2+‐sensitive pathway. The timing and Ca2+ sensitivity suggest a role in the primary signal transduction events of fertilization.
A plant metallothionein produced in E. coli
Authors: Kille Peter , Winge Dennis R. , Harwood John L. , Kay John
Published: October 2002

A metallothionein cDNA was generated from pea (Pisum sativum L.) roots, amplified by PCR and inserted into a plasmid for expression in E. coli. Purification of the resultant product generated 3 pools of cadmium‐containing material after DEAE‐cellulose chromatography. The amino acid composition of each was in excellent agreement with that predicted for pea metallothionein. A cadmium content of ∼6 g.atoms per mole of protein was estimated. N‐terminal sequence analysis revealed that the recombinant molecule had been proteolysed within the extended region linking the 2 cysteine‐rich (putative) metal‐binding regions. The significance of these findings in terms of the protein folding/targeting of the molecule are considered.
Isolation of cDNAs encoding a novel member of the neurotransmitter transporter gene family
Authors: Mayser Wolfgang , Betz Heinrich , Schloss Patrick
Published: October 2002

Degenerate oligonucleotides deduced from two regions of the rat γ‐aminobutyric acid (GABA) transporter were used to amplify related sequences from rat spinal cord by polymerase chain reaction (PCR). The resulting product then allowed isolation of two overlapping cDNA clones with an open reading frame encoding a hydrophobic polypeptide of 630 amino acids. This protein, termed NTT, exhibits 38% sequence homology to the GABA and 48% to the noradrenaline transporters and contains 12 putative transmembrane regions. PCR amplification revealed low‐level expression of NTT transcripts in spinal cord, but not in brain and cerebellum.