bluuebunny/crossref_metadata_2025 huggingface dataset:
Fas ligand expressed in colon cancer is not associated with increased apoptosis of tumor cells in vivo
Authors: Houston Aileen , Waldron‐Lynch Frank D. , Bennett Michael W. , Roche Desmond , O'Sullivan Gerald C. , Shanahan Fergus , O'Connell Joe
Published: August 2003

Expression of Fas ligand (FasL/CD95L) may help to maintain colon cancers in a state of immune privilege by inducing apoptosis of antitumor immune effector cells. Colon tumor‐derived cell lines appear to be relatively insensitive to apoptosis mediated by their own or exogenous FasL in vitro, despite expression of cell surface Fas. In our present study, we sought to investigate if FasL upregulated in human colon cancers leads to any increase in apoptosis of the tumor cells in vivo. FasL and Fas receptor (APO‐1/CD95) expression by tumor cells were detected immunohistochemically. Apoptotic tumor cell death was detected by immunohistochemistry for caspase‐cleaved cytokeratin‐18. FasL expression did not correlate with the extent of apoptosis of tumor cells. There was no significant local difference in the frequency of apoptosis of tumor cells between tumor nests that expressed FasL (mean = 2.4%) relative to those that did not (mean = 2.6%) (p = 0.625, n = 10; Wilcoxon signed rank). FasL expressed by the tumor cells appeared to be functional, since FasL expression in tumor nests correlated with diminished infiltration of tumor‐infiltrating lymphocytes (TILs). TILs were detected using immunohistochemistry for CD45. Expression of FasL by tumor nests was associated with a mean 4‐fold fewer TILs relative to FasL‐negative nests (range 2.4–33‐fold, n = 10, p < 0.003). Together, our results indicate that colon tumors are insensitive to FasL‐mediated apoptosis in vivo. © 2003 Wiley‐Liss, Inc.
Dual variant of Epstein‐Barr virus in Hodgkin/Reed‐Sternberg cells: Single‐cell PCR study on latent membrane protein‐1 gene
Authors: Kim Lian‐hua , Peh Suat‐cheng , Poppema Sibrand
Published: August 2003

Isolation of single cells permits analysis of DNA or RNA from individual cells among heterogeneous populations. This technique is particularly useful in the study of classical Hodgkin's lymphoma (cHL) due to the scarcity of H/RS tumor cells among large numbers of reactive leukocytes. In a previous study, we found a high frequency of dual LMP‐1 variant (concurrent presence of deleted and nondeleted variants) in cHL from whole‐tissue sections. For the present study, we applied a single‐cell isolation technique to determine the LMP‐1 oncogene variant in EBV‐associated H/RS cells. Five cases of EBV‐infected cHL, containing nondeleted (n = 1), deleted (n = 1) and dual infection (n =3) based on whole‐tissue section analysis, were selected for study. Paraffin‐embedded tissue sections were stained with antibody to LMP‐1 and positively stained H/RS cells isolated using a semiautomated micromanipulator. Each isolated single cell was subjected to PCR for amplification of the LMP‐1 gene flanking the 30 bp deletion region and Xho1 restriction site. Cases with either nondeleted variant or the deleted variant showed similar LMP‐1 variant expression in isolated single H/RS cells. However, 1 of the 3 cases with dual variants showed only the deleted variant in H/RS cells. The other 2 cases showed mixed patterns of deleted, nondeleted and dual LMP‐1 variants in isolated single H/RS cells. All cases showed loss of the Xho1 restriction site, with the exception of the case with nondeleted LMP‐1. Results of single‐H/RS cell analysis of the Xho1 restriction site concur with those of whole‐tissue section amplification. A mixed pattern of LMP‐1 variants was observed in isolated H/RS cells, and it is speculated that this is due to the accumulation of mutation and deletion events. © 2003 Wiley‐Liss, Inc.
Adaptation to the land: The skin of reptiles in comparison to that of amphibians and endotherm amniotes
Authors: Alibardi Lorenzo
Published: August 2003

The adaptation to land from amphibians to amniotes was accompanied by drastic changes of the integument, some of which might be reconstructed by studying the formation of the stratum corneum during embryogenesis. As the first amniotes were reptiles, the present review focuses on past and recent information on the evolution of reptilian epidermis and the stratum corneum. We aim to generalize the discussion on the evolution of the skin in amniotes. Corneous cell envelopes were absent in fish, and first appeared in adult amphibian epidermis. Stem reptiles evolved a multilayered stratum corneum based on a programmed cell death, intensified the production of matrix proteins (e.g., HRPs), corneous cell envelope proteins (e.g., loricrine‐like, sciellin‐like, and transglutaminase), and complex lipids to limit water loss. Other proteins were later produced in association to the soft or hairy epidermis in therapsids (e.g., involucrin, profilaggrin‐filaggrin, trichohyalin, trichocytic keratins), or to the hard keratin of hairs, quills, horns, claws (e.g., tyrosine‐rich, glycine‐rich, sulphur‐rich matrix proteins). In sauropsids special proteins associated to hard keratinization in scales (e.g., scale beta‐keratins, cytokeratin associated proteins) or feathers (feather beta‐keratins and HRPs) were originated. The temporal deposition of beta‐keratin in lepidosaurian reptiles originated a vertical stratified epidermis and an intraepidermal shedding layer. The evolutions of the horny layer in Therapsids (mammals) and Saurospids (reptiles and birds) are discussed. The study of the molecules involved in the dermo‐epidermal interactions in reptilian skin and the molecular biology of epidermal proteins are among the most urgent future areas of research in the biology of reptilian skin. J. Exp. Zool. (Mol. Dev. Evol.) 298B:12–41, 2003. © 2003 Wiley‐Liss, Inc.
Avian skin development and the evolutionary origin of feathers
Authors: Sawyer Roger H. , Knapp Loren W.
Published: August 2003

The discovery of several dinosaurs with filamentous integumentary appendages of different morphologies has stimulated models for the evolutionary origin of feathers. In order to understand these models, knowledge of the development of the avian integument must be put into an evolutionary context. Thus, we present a review of avian scale and feather development, which summarizes the morphogenetic events involved, as well as the expression of the beta (β) keratin multigene family that characterizes the epidermal appendages of reptiles and birds. First we review information on the evolution of the ectodermal epidermis and its beta (β) keratins. Then we examine the morphogenesis of scutate scales and feathers including studies in which the extraembryonic ectoderm of the chorion is used to examine dermal induction. We also present studies on the scaleless (sc) mutant, and, because of the recent discovery of “four‐winged” dinosaurs, we review earlier studies of a chicken strain, Silkie, that expresses ptilopody (pti), “feathered feet.” We conclude that the ability of the ectodermal epidermis to generate discrete cell populations capable of forming functional structural elements consisting of specific members of the β keratin multigene family was a plesiomorphic feature of the archosaurian ancestor of crocodilians and birds. Evidence suggests that the discrete epidermal lineages that make up the embryonic feather filament of extant birds are homologous with similar embryonic lineages of the developing scutate scales of birds and the scales of alligators. We believe that the early expression of conserved signaling modules in the embryonic skin of the avian ancestor led to the early morphogenesis of the embryonic feather filament, with its periderm, sheath, and barb ridge lineages forming the first protofeather. Invagination of the epidermis of the protofeather led to formation of the follicle providing for feather renewal and diversification. The observations that scale formation in birds involves an inhibition of feather formation coupled with observations on the feathered feet of the scaleless (High‐line) and Silkie strains support the view that the ancestor of modern birds may have had feathered hind limbs similar to those recently discovered in nonavian dromaeosaurids. And finally, our recent observation on the bristles of the wild turkey beard raises the possibility that similar integumentary appendages may have adorned nonavian dinosaurs, and thus all filamentous integumentary appendages may not be homologous to modern feathers. J. Exp. Zool. (Mol. Dev. Evol.) 298B:57–72, 2003. © 2003 Wiley‐Liss, Inc.
Molecular biology of feather morphogenesis: A testable model for evo‐devo research
Authors: Widelitz Randall B. , Jiang Ting Xin , Yu Mingke , Shen Ted , Shen Jen‐Yee , Wu Ping , Yu Zhicao , Chuong Cheng‐Ming
Published: August 2003

Darwin's theory describes the principles that are responsible for evolutionary change of organisms and their attributes. The actual mechanisms, however, need to be studied for each species and each organ separately. Here we have investigated the mechanisms underlying these principles in the avian feather. Feathers comprise one of the most complex and diverse epidermal organs as demonstrated by their shape, size, patterned arrangement and pigmentation. Variations can occur at several steps along each level of organization, leading to highly diverse forms and functions. Feathers develop gradually during ontogeny through a series of steps that may correspond to the evolutionary steps that were taken during the phylogeny from a reptilian ancestor to birds. These developmental steps include 1) the formation of feather tract fields on the skin surfaces; 2) periodic patterning of the individual feather primordia within the feather tract fields; 3) feather bud morphogenesis establishing anterio ‐ posterior (along the cranio ‐ caudal axis) and proximo ‐ distal axes; 4) branching morphogenesis to create the rachis, barbs and barbules within a feather bud; and 5) gradual modulations of these basic morphological parameters within a single feather or across a feather tract. Thus, possibilities for variation in form and function of feathers occur at every developmental step. In this paper, principles guiding feather tract formation, distributions of individual feathers within the tracts and variations in feather forms are discussed at a cellular and molecular level. J. Exp. Zool. (Mol. Dev. Evol.) 298B:109–122, 2003. © 2003 Wiley‐Liss, Inc.
Horse hooves and bird feathers: Two model systems for studying the structure and development of highly adapted integumentary accessory organs—the role of the dermo‐epidermal interface for the micro‐architecture of complex epidermal structures
Authors: Bragulla Hermann , Hirschberg Ruth M.
Published: August 2003

Accessory organs of the integument are locally modified parts of the potentially feather‐bearing skin in birds (e.g., the rhamphotheca, claws, or scales), and of the potentially hairy skin in mammals (e.g., the rhinarium, nails, claws, or hooves). These special parts of the integument are characterised by a modified structure of their epidermal, dermal and subcutaneous layers. The developmental processes of these various integumentary structures in birds and mammals show both similarities and differences. For example, the development of the specialised epidermal structures of both feathers and the hoof capsule is influenced by the local three‐dimensional configuration of the dermis. However, in feathers, in contrast to hooves, the arrangement of the corneous cells is only partially a direct result of the particular arrangement and shape of the dermal surface of the papillary body. Whereas the diameter of the feather papilla, as well as the number, length, and width of dermal ridges on the surface of the feather papilla influence the three‐dimensional architecture of the feather rami, there is no apparent direct correlation between the dermo‐epidermal interface and the development of the highly ordered architecture of the radii and hamuli in the feather vane. In order to elucidate this morphogenic problem and the problem of locally different processes of keratinisation and cornification, the structure and development of feathers in birds are compared to those of the hoof capsule in horses. The equine hoof is the most complex mammalian integumentary structure, which is determined directly by the dermal surface of the papillary body. Perspectives for further research on the development of modified integumentary structures, such as the role of the dermal microangioarchitecture and the selective adhesion and various differentiation pathways of epidermal cells, are discussed. J. Exp. Zool. (Mol. Dev. Evol.) 298B:140–151, 2003. © 2003 Wiley‐Liss, Inc.
Long‐term evaluation of retinal function in Prph2Rd2/Rd2 mice following AAV‐mediated gene replacement therapy
Authors: Schlichtenbrede Frank C. , da Cruz Lyndon , Stephens Clare , Smith Alexander J. , Georgiadis Anastasios , Thrasher Adrian J. , Bainbridge James W. B. , Seeliger Mathias W. , Ali Robin R.
Published: August 2003

Background

Prph2Rd2/Rd2 mice have a retinal degeneration due to a null mutation for the Prph2 gene which encodes a photoreceptor‐specific glycoprotein, peripherin2, essential for outer‐segment formation. We have previously shown improvement of photoreceptor function at a single time point following AAV‐mediated gene replacement therapy. Here we quantify the functional rescue over a 15‐week time course and present a detailed analysis of the improvement in retinal function.

Methods

An AAV2 vector, AAV.rho.rds, carrying a peripherin2 c‐DNA, was in injected subretinally into 10‐day‐old Prph2Rd2/Rd2 mice. One group was injected at a single time point while in a second group the injections were repeated after 5 days. The effect of treatment was analysed histologically using electron microscopy and electroretinography (ERG) was used to assess functional changes. Treated mice were recorded at regular intervals over 15 weeks. Untreated contralateral eyes served as internal control.

Results

A significant increase in b‐wave amplitude was first noted 3 weeks after treatment of 10‐day‐old Prph2Rd2/Rd2 mice and persisted for up to 14 weeks. An increase in the area of retina exposed to vector resulted in a significant increase in both b‐wave amplitude and persistence.

Conclusions

In this study AAV‐mediated gene replacement in Prph2Rd2/Rd2 mice resulted in a significant functional improvement over a period of 14 weeks. These results support the utility of gene therapy approaches as treatment for photoreceptor dystrophies. Copyright © 2003 John Wiley & Sons, Ltd.
Failure to generate atheroprotective apolipoprotein AI phenotypes using synthetic RNA/DNA oligonucleotides (chimeraplasts)
Authors: Manzano Anna , Mohri Zahra , Sperber Galia , Ogris Manfred , Graham Ian , Dickson George , Owen James S.
Published: August 2003

Background

Elevated plasma high‐density lipoprotein (HDL), and its major constituent apolipoprotein AI (apoAI), are cardioprotective. Paradoxically, two natural variants of apoAI, termed apoAIMilano and apoAIParis, are associated with low HDL, but nevertheless provide remarkable protection against heart disease for heterozygous carriers and may even lead to longevity. Both variants arise from point mutations and have Arg173 and Arg151 to Cys substitutions, respectively, which allow disulphide‐linked dimers to form. Potentially, synthetic RNA/DNA oligonucleotides (chimeraplasts) can permanently correct single point mutations in genomic DNA. Here, we use a variation of such targeted gene repair technology, ‘gain‐of‐function chimeraplasty’, and attempt to enhance the biological activity of apoAI by altering a single genomic base to generate the atheroprotective phenotypes, apoAIMilano and apoAIParis.

Methods

We targeted two cultured cell lines that secrete human apoAI, hepatoblastoma HepG2 cells and recombinant CHO‐AI cells, using standard 68‐mer chimeraplasts with polyethyleneimine (PEI) as carrier and then systematically varied several experimental conditions. As a positive control we targeted the dysfunctional APOE2 gene, which we have previously converted to wild‐type APOE3.

Results

Conversion of wild‐type apoAI to apoAIMilano proved refractory, with limited correction in CHO‐AI cells only. However, a successful conversion to apoAIParis was achieved, as demonstrated by polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP) analysis and direct genomic sequencing. Unexpectedly, attempts with a new batch of 68‐mer chimeraplast to enhance conversion, by using different delivery vehicles, including chemically modified PEI, failed to show a base change; nor could conversion be detected with an 80‐mer or a 52–76‐mer series. In contrast, when a co‐culture of CHO‐E2 and CHO‐AI cells was co‐targeted, a clear conversion of apoE2 to apoE3 was seen, whereas no apoAIParis could be detected. When the individual chimeraplasts were analysed by denaturing electrophoresis only the active apoE2‐to‐E3 chimeraplast gave a sharp band.

Conclusions

Our findings suggest that different batches of chimeraplasts have variable characteristics and that their quality may be a key factor for efficient targeting and/or base conversion. We conclude that, although an evolving technology with enormous potential, chimeraplast‐directed gene repair remains problematical. Copyright © 2003 John Wiley & Sons, Ltd.
A bicistronic SIN‐lentiviral vector containing G156A MGMT allows selection and metabolic correction of hematopoietic protoporphyric cell lines
Authors: Richard Emmanuel , Géronimi Fabien , Lalanne Magalie , Ged Cécile , Redonnet‐Vernhet Isabelle , Lamrissi‐Garcia Isabelle , Gerson Stanton L , de Verneuil Hubert , Moreau‐Gaudry François
Published: August 2003

Background

Erythropoietic protoporphyria (EPP) is an inherited disease characterised by a ferrochelatase (FECH) deficiency, the latest enzyme of the heme biosynthetic pathway, leading to the accumulation of toxic protoporphyrin in the liver, bone marrow and spleen. We have previously shown that a successful gene therapy of a murine model of the disease was possible with lentiviral vectors even in the absence of preselection of corrected cells, but lethal irradiation of the recipient was necessary to obtain an efficient bone marrow engraftment. To overcome a preconditioning regimen, a selective growth advantage has to be conferred to the corrected cells.

Methods

We have developed a novel bicistronic lentiviral vector that contains the human alkylating drug resistance mutant O6‐methylguanine DNA methyltransferase (MGMT G156A) and FECH cDNAs. We tested their capacity to protect hematopoietic cell lines efficiently from alkylating drug toxicity and correct enzymatic deficiency.

Results

EPP lymphoblastoid (LB) cell lines, K562 and cord‐blood‐derived CD34+ cells were transduced at a low multiplicity of infection (MOI) with the bicistronic constructs. Resistance to O6‐benzylguanine (BG)/N,N′‐bis(2‐chloroethyl)‐N‐nitrosourea (BCNU) was clearly shown in transduced cells, leading to the survival and expansion of provirus‐containing cells. Corrected EPP LB cells were selectively amplified, leading to complete restoration of enzymatic activity and the absence of protoporphyrin accumulation.

Conclusions

This study demonstrates that a lentiviral vector including therapeutic and G156A MGMT genes followed by BG/BCNU exposure can lead to a full metabolic correction of deficient cells. This vector might form the basis of new EPP mouse gene therapy protocols without a preconditioning regimen followed by in vivo selection of corrected hematopoietic stem cells. Copyright © 2003 John Wiley & Sons, Ltd.
Electrophysiologically “complex” glial cells freshly isolated from the hippocampus are immunopositive for the chondroitin sulfate proteoglycan NG2
Authors: Schools Gary P. , Zhou Min , Kimelberg Harold K.
Published: August 2003

We have recently described a subgroup of isolated glial fibrillary acidic protein‐positive (GFAP+) hippocampal astrocytes that predominantly express outwardly rectifying currents (which we term “ORAs” for outwardly rectifying astrocytes), which are similar to the currents already described for hippocampal GFAP− “complex glia.” We now report that post‐recording staining of cells that were first selected as “complex” by morphology and then confirmed by their electrophysiological characteristics were NG2+ ∼90% of the time. Also, the morphology of freshly isolated NG2+ cells differs from that of isolated GFAP+ ORAs in having a smaller and round cell body with thinner processes, which usually are collapsed back onto the soma. Upon detailed examination, NG2+ cells were found to differ quantitatively in some electrophysiological characteristics from GFAP+ ORAs. The outward, transient K+ currents (IKa) in the NG2+ cells showed a slower decay than the IKa in ORAs, and their density decreased in NG2+ cells from older animals. The other two major cation currents, the voltage‐activated Na+ and outwardly delayed rectifier K+ currents, were similar in NG2+ cells and ORAs. To further distinguish isolated complex cells from outwardly rectifying GFAP+ astrocytes, we performed immunocytochemistry for glial markers in fixed, freshly isolated rat hippocampal glia. NG2+ cells were negative for GFAP and also for the astrocytic glutamate transporters GLT‐1 and GLAST. Thus the isolated hippocampal NG2+ glial cells, though having an electrophysiological phenotype similar to that of ORAs, are an immunologically and morphologically distinct glial cell population and most likely represent NG2+ cells in situ. © 2003 Wiley‐Liss, Inc.
Bcl‐2 deficiency deprives peripheral nerves of neurotrophic activity against injured optic nerve
Authors: Kotulska Katarzyna , Lewin‐Kowalik Joanna , Larysz‐Brysz Magdalena , Marcol Wiesław , Fus Zbigniew
Published: August 2003

Optic nerve injury leads to retinal ganglion cell apoptosis, thus preventing fiber regeneration. Peripheral nerve grafts are known to promote survival and regeneration in injured adult mammalian central nervous system, including optic nerve, but the mechanisms of their activity remain unclear. It is likely that they attenuate the apoptotic cascade triggered by axotomy in retinal ganglion cells. The aim of this work was to examine the role of the antiapoptotic gene bcl‐2 in the optic nerve regeneration induced by such grafts. Experiments were carried out on bcl‐2‐deficient and wild‐type mice. We have reported previously that predegeneration markedly enhances neurotrophic activity of peripheral nerve grafts, so we applied both predegenerated and non‐predegenerated implants to the transected optic nerves. We studied the neurotrophic effects of bcl‐2‐deficient grafts on wild‐type and bcl‐2 knock‐out optic nerves, as well as wild‐type grafts on both strains of mouse optic nerves. After application of fluorescent dye to the end of the graft, we counted the stained retinal ganglion cells. Predegenerated wild‐type grafts promoted survival and outgrowth of retinal ganglion cells axons in both types of mice. By contrast, non‐predegenerated and predegenerated bcl‐2‐deficient grafts induced little or no regeneration in the optic nerves. These results indicate that the lack of bcl‐2 gene does not deprive retinal ganglion cells of their regenerative potential. At the same time, we found that bcl‐2 knock‐out dispossesses peripheral nerves of their neurotrophic activity. © 2003 Wiley‐Liss, Inc.
Increased turnover of Na‐K ATPase molecules in rat brain after rapid eye movement sleep deprivation
Authors: Majumdar Sudipta , Faisal Mohd , Madan Vibha , Mallick Birendra N.
Published: August 2003

It has been shown that rapid eye movement (REM) sleep deprivation increases Na‐K ATPase activity. Based on kinetic study, it was proposed that increased activity was due to enhanced turnover of enzyme molecules. To test this, anti‐α1 Na‐K ATPase monoclonal antibody (mAb 9A7) was used to label Na‐K ATPase molecules. These labeled enzymes were quantified on neuronal membrane by two methods: histochemically on neurons in tissue sections from different brain areas, and by Western blot analysis in control and REM sleep‐deprived rat brains. The specific enzyme activity was also estimated and found to be increased, as in previous studies. The results confirmed our hypothesis that after REM sleep deprivation, increased Na‐K ATPase activity was at least partly due to increased turnover of Na‐K ATPase molecules in the rat brain. © 2003 Wiley‐Liss, Inc.
Zebrafish Cx35: Cloning and characterization of a gap junction gene highly expressed in the retina
Authors: McLachlan Elizabeth , White Thomas W. , Ugonabo Chioma , Olson Carl , Nagy James I. , Valdimarsson Gunnar
Published: August 2003

The vertebrate connexin gene family encodes protein subunits of gap junction channels, which provide a route for direct intercellular communication. Consequently, gap junctions play a vital role in many developmental and homeostatic processes. Aberrant functioning of gap junctions is implicated in many human diseases. Zebrafish are an ideal vertebrate model to study development of the visual system as they produce transparent embryos that develop rapidly, thereby facilitating morphological and behavioral testing. In this study, zebrafish connexin35 has been cloned from a P1 artificial chromosome (PAC) library. Sequence analysis shows a high degree of similarity to the Cx35/36 orthologous group, which are expressed primarily in nervous tissue, including the retina. The gene encodes a 304‐amino acid protein with a predicted molecular weight of approximately 35 kDa. Injection of zebrafish Cx35 RNA into paired Xenopus oocytes elicited intercellular electrical coupling with weak voltage sensitivity. In development, Cx35 is first detectable by Northern analysis and RT‐PCR, at 2 days post‐fertilization (2 dpf), and in the adult it is expressed in the brain and retina. Immunohistochemical analysis revealed that the Cx35 protein is expressed in two sublaminae of the inner plexiform layer of the adult retina. A similar pattern was seen in the 4 and 5 dpf retina, but no labeling was detected in the retina of earlier embryos. © 2003 Wiley‐Liss, Inc.
Characterization of activated retinal microglia following optic axotomy
Authors: Zhang Cheng , Tso Mark O.M.
Published: August 2003

Microglia are prominently involved in neural degenerative diseases of the CNS and the retina. In this study, we determined the activation and phagocytotic function of different subtypes of retinal microglial cells at 1 week and 1 month following optic axotomy. Fluorescent DiI crystals were placed at the stumps of the cut optic nerves of Lewis rats to retrolabel retinal ganglion cells. Microglial cells were indirectly labeled as they phagocytosed the dye particles in the dying ganglion cells. OX‐42, 5D4, ED1, and OX‐6 antibodies were used for immunohistochemical study. The OX‐42‐ and 5D4‐positive microglial cells were increased in the inner retinal layers after optic axotomy. The increase of OX‐42‐positive cells was considerably greater than that of 5D4‐positive cells. The 5D4‐positive cells were ramified in shape, whereas OX‐42‐positive cells were ameboid and ovoid. Both 5D4‐ and OX‐42‐positive cells phagocytosed dying ganglion cells at 1 week and 1 month after axotomy. Scattered ameboid ED1‐positive cells were detected in the normal retina and showed phagocytotic activity at 1 month after optic axotomy. The number of ED1‐positive cells in the retina was unchanged after axotomy. In optic axotomy, three types of microglial cells were activated, namely, 5D4‐positive ramified cells and OX‐42‐ and ED1‐positive ameboid cells. All of them exhibited the phagocytosis of dying ganglion cells. Insofar as the blood–retinal barrier presumably remained intact in optic axotomy, the OX‐42‐ and 5D4‐positive cells might derive from resident microglial cells. The ED1‐positive cells, presumably recently blood‐borne macrophage in the CNS, remained the same number in the axotomized retina. © 2003 Wiley‐Liss, Inc.
Transcription factor activator protein‐1 expressed by kainate treatment can bind to the non‐coding region of mitochondrial genome in murine hippocampus
Authors: Ogita Kiyokazu , Fujinami Yoshiaki , Kitano Masahiro , Yoneda Yukio
Published: August 2003

We have demonstrated previously that the transcription factor activator protein‐1 (AP‐1) complex is translocated into mitochondria into the nucleus in murine hippocampus after systemic kainate injection (Ogita et al. [2002] J. Neurosci. 22:2561–2570). The present study investigates whether the mitochondrial AP‐1 complex translocated in response to kainate treatment binds to AP‐1‐like sites located at the non‐coding region of the mitochondrial genome in mouse hippocampus. There are 10 sites with sequences similar to the nuclear AP‐1 site in the non‐coding region. Of 10 pieces (MT‐1–MT‐10) of synthesized double‐stranded oligonucleotides, each containing a mitochondrial AP‐1‐like site, MT‐3, MT‐4, and MT‐9 were effective in inhibiting mitochondrial AP‐1 DNA binding enhanced by kainate. Electrophoresis mobility shift analysis using radiolabeled MT‐3 and MT‐9 probes demonstrated marked enhancement with binding of these 2 probes in hippocampal mitochondrial extracts prepared 2–6 hr after kainate treatment. Unlabeled AP‐1 probe was more potent than unlabeled MT‐9 probe in inhibiting the mitochondrial MT‐9 binding. Supershift analysis revealed participation of particular Fos/Jun family proteins, such as c‐Fos, Fos‐B, c‐Jun, Jun‐B, and Jun‐D, in MT‐9 binding in hippocampal mitochondrial extracts prepared 4 hr after kainate treatment. Immunoprecipitation analysis using anti‐c‐Fos antibody demonstrated that c‐Fos associated with the mitochondrial genome in hippocampal mitochondria prepared from kainate‐treated animals. These results suggest that the AP‐1 complex expressed by in vivo kainate treatment would bind to AP‐1‐like sites in the non‐coding region of the mitochondrial genome after translocation into mitochondria from murine hippocampus. © 2003 Wiley‐Liss, Inc.
Increases in matrix metalloproteinase‐9 and tissue inhibitor of matrix metalloproteinase‐1 mRNA after cerebral contusion and depolarisation
Authors: von Gertten Christina , Holmin Staffan , Mathiesen Tiit , Sandberg Nordqvist Ann‐Christin
Published: August 2003

Matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) play major roles in physiological extracellular matrix turnover during normal development and in pathological processes. In brain, increases in MMP activity occur, for example, in multiple sclerosis, Alzheimer's disease, and after head trauma. We examined MMP‐9 and TIMP‐1, ‐2, and ‐3 in events after head trauma. A time‐course study was carried out using two different rat injury models, cerebral contusion and depolarisation. Brains were analysed by RT‐PCR and in situ hybridisation. We observed a distinct and time‐dependent upregulation of MMP‐9 and TIMP‐1 mRNA in ipsilateral cortical areas. MMP‐9 mRNA levels were upregulated 1 day after cerebral contusion with a peak at Day 4. Depolarisation per se, which also occurs after traumatic brain injury, lead to delayed increase of MMP‐9 mRNA, 4 days post application. At Day 14, MMP‐9 mRNA levels were indistinguishable from controls in both models. TIMP‐1 mRNA increases were observed in both models 4 hr after injury, and increased further at Days 1 and 4. At Day 14, mRNA levels declined and were no higher than control levels. No alterations in mRNA levels were noted for TIMP‐2 or ‐3. Our results support earlier reports on MMP‐9 involvement in brain injury. It also shows a role for TIMP‐1 in the mechanisms of trauma, where depolarisation could be the mechanism responsible for this upregulation. © 2003 Wiley‐Liss, Inc.
Optimization of hot water extraction of roselle juice using response surface methodology: a comparative study with other extraction methods
Authors: Wong Peng‐Kong , Yusof Salmah , Ghazali Hasanah Mohd , Che Man Yaakob bin
Published: August 2003

Roselle has regained the attention of many fruit juice manufacturers in Malaysia as a product that contains high ascorbic acid and anthocyanins. However, ascorbic acid and anthocyanin pigments can be easily destroyed during processing of fruit juice. Therefore, in the present study, the effects of different processing methods, namely hot water extraction (HWE), hot water blending, cold water blending and screw press, on the changes in anthocyanins and ascorbic acid contents of roselle juice were evaluated. The anthocyanins and ascorbic acid contents of roselle were determined using the pH differential method and high‐performance liquid chromatography respectively. The physico‐chemical characteristics and sensory properties of roselle juice were also evaluated. The results of the study indicate that the HWE method is the most effective extraction method, resulting in high anthocyanins and ascorbic acid contents of 43 g l−1 (as delphinidin‐3‐glucoside) and 2.34 g kg−1 respectively. The optimum juice extraction conditions were 3.5 h at 60 °C. Copyright © 2003 Society of Chemical Industry
Soil moisture stress‐induced alterations in bioconstituents determining tea quality
Authors: Jeyaramraja PR , Pius PK , Raj Kumar R , Jayakumar D
Published: August 2003

The impact of water stress on the biochemical constituents that determine black tea quality was investigated. Phenylalanine ammonia lyase (PAL) activity was highest in the drought tolerant ‘Assam’ cultivar UPASI‐2, followed by UPASI‐8 and UPASI‐9, under non‐stress conditions. Under soil moisture stress a reduction in PAL activity was found in all three clones investigated. A strong positive correlation was observed between an increase in soil moisture deficit and a decrease in PAL activity. Lower PAL activity correlated well with lower synthesis of flavanols such as epigallocatechin gallate (EGCG) and epicatechin gallate (ECG), which are important precursors of theaflavin‐3,3′‐digallate that determines final tea quality. Altered synthesis of EGCG and ECG could be due to their molecular rearrangement at elevated leaf temperature during drought. Synthesis of quality constituents such as gallic acid and caffeine declined significantly owing to both drought and waterlogging stress. The reduction in gallic acid due to water stress could lead to lower synthesis of theaflavin fractions such as epitheaflavic acid, epitheaflavic acid‐3′‐gallate and theaflavic acid and, thereby, quality deterioration. Similarly to drought, flooding stress was also found to alter the biochemical constituents necessary for tea quality. Copyright © 2003 Society of Chemical Industry
Modified method for the determination of pyruvic acid with dinitrophenylhydrazine in the assessment of onion pungency
Authors: Anthon Gordon E , Barrett Diane M
Published: August 2003

Modifications to the commonly used Schwimmer and Weston procedure for determining pyruvic acid in onions are proposed. These modifications involve changes in the concentrations and volumes of the reagents and the use of 515 rather than 420 nm as the wavelength for measurements. These changes improve both the linearity and the sensitivity of the assay and eliminate interference from coloured material found in yellow onions. With these changes, more reliable determinations of pyruvic acid and thus of onion pungency should be possible. Copyright © 2003 Society of Chemical Industry
Removing τ‐fluvalinate residues from press‐extracted honey
Authors: Waliszewski Stefan M , Infanzón Rosa M , Carvajal Octavio , Maxwell Hart Mary
Published: August 2003

Honey is obtained from the hives of European honeybees. Honeybees can be infested by Varroa parasitism, which affects honey production. The acaricide τ‐fluvalinate used in beehives to control Varroa mites, can leave its residues in honey, pollen and wax. A monitoring study was undertaken to determine the τ‐fluvalinate residues in honey samples taken from treated hives. The τ‐fluvalinate residues were determined by gas chromatography using a short, non‐polar packed column with electron capture detection. Analysis of 50 samples showed an average residue level of 15 µg kg−1. The same samples were filtered through Whatman filter paper to separate the honey from residual suspended particles. The filtered honeys showed no acaricide residues above the detection limit of 3 µg kg −1, while the average residue level determined in the sediments was 392 µg kg −1. Thus the residues of τ‐fluvalinate in honey can be eliminated by separating the honey from residual suspended particles that contain acaricide residues. Copyright © 2003 Society of Chemical Industry
Antioxidant capacity and phenolic content in blueberries as affected by genotype and growing season
Authors: Howard Luke R , Clark John R , Brownmiller Cindi
Published: August 2003

Blueberry fruits from five commercial cultivars and 13 breeding selections grown at the same locations over two growing seasons were analysed for total phenolics (TPH), total anthocyanins (ACY), total hydroxycinnamic acids (HCA), total flavonols (FLA), fruit weight and oxygen radical‐absorbing capacity (ORAC). Variation in ORAC, TPH, ACY, HCA, FLA and fruit weight among genotypes was much greater than that observed between growing seasons, indicating that genetics plays a more important role than growing season in influencing ORAC and phenolic content in blueberries. Significant main effects for growing season and genotype × growing season observed for ORAC, ACY, HCA and fruit weight demonstrate that environmental growing conditions can impact levels of phenolics and ORAC in blueberries and that certain genotypes vary in their capacity to synthesise phenolics under different growing conditions. In general, genotypes with smaller berries had higher ORAC values and levels of TPH, ACY, HCA and FLA than large‐berried genotypes. Over both growing seasons, ORAC correlated highly with TPH, ACY, HCA and FLA, while fruit weight correlated inversely with all phenolics measured. Our results indicate that blueberry genotypes should be screened over multiple growing seasons in order to identify antioxidant‐ and phenolic‐rich germplasm. Copyright © 2003 Society of Chemical Industry
Decreased expression of human papillomavirus E2 protein and transforming growth factor‐β1 in human cervical neoplasia as an early marker in carcinogenesis
Authors: Torng Pao‐Ling , Chan Wing‐Yee , Lin Chin‐Tang , Huang Su‐Cheng
Published: August 2003

Background and Objectives

Human papillomavirus (HPV) is thought to be one of the possible causative factors in cervical carcinogenesis, and cervical carcinoma cells are refractory to tumor transforming growth factor (TGF)‐β1. The purpose of this study is to investigate the possible cause–effect association between HPV and TGF‐β1 during cervical tumorigenesis.

Methods

We assessed the expression of HPV capsid proteins, HPV‐16 E7, HPV‐16 E2 (C and N terminals), TGF‐β1, and their receptors TGF‐β RI and RII by immunohistochemistry in 48 paraffin‐embedded blocks of tumor tissue derived from patients of cervical neoplasia.

Results

Expression of TGF‐β1 decreased as tumor cells progressed from cervical intraepithelial neoplasia (CIN)1, CIN2, CIN3, to microinvasive carcinoma (P < 0.05). Levels of TGF‐βRI and TGFβ‐RII stayed the same in all cases. HPV was found in 89.6% of the studied sections, and cervical lesions without HPV infection expressed significantly less TGF‐β1 (P < 0.05). By comparing the expression pattern of TGF‐β1 and HPV in the neoplastic cells with that of normal cervical epithelium in each section, we found loss of HPV‐16 E2 higher in CIN3 (15/24) than in CIN1 or CIN2 (3/7), and there is a significant trend that loss of HPV‐16 E2 expression correlated with a >50% loss of TGF‐β1 at the lesion site (P < 0.05).

Conclusions

Our result showed co‐suppression of HPV and TGF‐β1 expression during progression of cervical squamous cell cancer. Using antibody against HPV‐16 E2 may be an auxiliary tool for the investigation of cervical tumor progression. J. Surg. Oncol. 2003;84:17–23. © 2003 Wiley‐Liss, Inc.
Primary malignant melanoma of the lip
Authors: Froix Anthony J. , Salti George I.
Published: August 2003

Melanoma of the lip is a very rare clinical entity. As such, no large studies are available to help in diagnosis and treatment. We report our results on 11 patients with lip melanoma. Patients with lip melanoma should be approached in a similar fashion to patients with other cutaneous melanomas. J. Surg. Oncol. 2003;84:7–9. © 2003 Wiley‐Liss, Inc.
Clinical benefit of chemosensitivity test for patients with regional lymph node–positive esophageal squamous cell carcinoma
Authors: Nakamori Mikihito , Iwahashi Makoto , Nakamura Masaki , Yamaue Hiroki
Published: August 2003

Background and Objectives

Patients who have undergone resection for lymph node–positive esophageal squamous cell carcinoma are at high risk of recurrence and early death. The role of the postoperative adjuvant chemotherapy in this population needs to be determined. The present study was designed to determine whether the chemosensitivity test in fresh human esophageal squamous cell carcinoma, using highly purified tumor cells, correlates with the clinical response.

Methods

A retrospective review of all patients with resected squamous cell carcinoma of the thoracic esophagus between 1993 and 2000 was performed. We determined the chemosensitivity for cisplatin (CDDP), 5‐fluorouracil (5‐FU), mitomycin C, and adriamycin in vitro in fresh human esophageal squamous cell carcinoma using the MTT assay. Regional lymph node–positive (N1) patients who received sequential postoperative chemotherapy were compared with lymph node positive patients who underwent surgery alone.

Results

A total of 50 patients were reviewed, and chemosensitivity tests were successfully performed in 46 patients: 20 patients received surgery alone (S group), and 26 patients received surgery plus postoperative chemotherapy (SC group) according to results of MTT assay using highly purified tumor cells. When the SC group was divided into an SC‐low group (inhibition rate of CDDP + 5‐FU was below 85%, n = 15) and an SC‐high group (over 85%, n = 11), the SC‐high group showed more significant survival prolongation than the S group or the SC‐low group (P < 0.01).

Conclusions

Our results suggest that the results of the conventional MTT assay may be useful in evaluating the optimum adjuvant chemotherapy for patients with regional lymph node positive (pN1) esophageal squamous cell carcinoma. J. Surg. Oncol. 2003;84:10–16. © 2003 Wiley‐Liss, Inc.
A new proliferation marker, minichromosome maintenance protein 2, is associated with tumor aggressiveness in esophageal squamous cell carcinoma
Authors: Kato Hiroyuki , Miyazaki Tatsuya , Fukai Yasuyuki , Nakajima Masanobu , Sohda Makoto , Takita Junko , Masuda Norihiro , Fukuchi Minoru , Manda Ryokuhei , Ojima Hitoshi , Tsukada Katsuhiko , Asao Takayuki , Kuwano Hiroyuki
Published: August 2003

Background

Our aim was to determine whether minichromosome maintenance protein (MCM) 2 expression is useful in predicting tumor proliferation rates and outcome after therapy in patients undergoing surgery for esophageal squamous cell carcinoma (SCC). In addition, we evaluated whether the expression of this proliferation marker was correlated with that of another marker, Ki‐67, in esophageal SCC.

Methods

We examined immunohistochemical expression of MCM2 and Ki‐67 in surgically resected tissues from 93 patients with esophageal SCC.

Results

The mean nuclear labeling index of MCM2 was markedly higher in SCC than in normal epithelia (P < 0.0001). A comparison of MCM2 labeling index and clinicopathological characteristics in 93 patients with esophageal cancer revealed significant associations between MCM2 labeling index and tumor status (P < 0.05), lymph node status (P < 0.01), metastatic status (P < 0.01), pathologic stage (P < 0.01), histologic grade (P < 0.05), and poor prognosis (P < 0.05). A comparison of Ki‐67 labeling index and clinicopathological characteristics revealed significant associations between Ki‐67 labeling index and lymph node status (P < 0.01), metastatic status (P < 0.05), pathologic stage (P < 0.05), histologic grade (P < 0.05), and poor prognosis (P < 0.05). The relationship between MCM2 and Ki‐67 labeling indices in the primary tumor showed a significant correlation (P < 0.05), but the MCM 2 labeling index was considerably higher.

Conclusions

MCM2 may be a more reliable and useful marker than Ki‐67 in assessing the growth of normal and tumor cells and in evaluating tumor aggressiveness and prognostic value in patients with esophageal SCC. J. Surg. Oncol. 2003;84:24–30. © 2003 Wiley‐Liss, Inc.
Treatment of oral lichen planus with the 308‐nm UVB excimer laser—early preliminary results in eight patients
Authors: Köllner Katja , Wimmershoff Monika , Landthaler Michael , Hohenleutner Ulrich
Published: August 2003

Background and Objectives

Oral lichen planus (OLP) is a chronic disease of uncertain origin. Many patients with OLP are refractory to all available therapies. The 308‐nm excimer laser was used as a possible additional method in the treatment of OLP.

Study Design/Materials and Methods

Eight patients with OLP were treated using the 308‐nm UVB excimer laser.

Results

Clinical improvement was achieved in six patients. Two patients showed complete remission, of which one patient showed recurrence of the lesions after 4 weeks.

Conclusion

The preliminary results in our opinion warrant further studies of this treatment. Lasers Surg. Med. 33:158–160, 2003. © 2003 Wiley‐Liss, Inc.
H‐NMR spin‐lattice and correlation times of burned soft‐tissue after treatment with an infrared pulsed laser device
Authors: Rodríguez‐Santana Elizabeth , Santana‐Blank Luis A. , Reyes Heberto , Santana‐Rodríguez Karin E. , Hunger Manfred , Orellana Rafael , Ortega Daniel
Published: August 2003

Background and Objectives

The aim of this study was to investigate the spin‐lattice (T1, 1/T1) and correlation times (τc) of burned soft‐tissue after treatment with an infrared (IR) pulsed laser device (IPLD, 904 nm pulsed at 3 MHz).

Study Design/Materials and Methods

Seven groups (GI–GVII), each consisting of four albino rats, were used. Groups I–VI were anesthetized and burned with a hot tip: GI, GIII, GV were not irradiated; GII, GIV, GVI were irradiated at 0; 0 and 24; and 0, 24, and 48 hours, respectively. A control group (GVII) was neither burned nor irradiated. Samples from all groups were evaluated using a 90 MHz hydrogen nuclear magnetic resonance (H‐NMR) spectrometer. An unpaired Student's t‐test and an ANOVA I were preformed (α = 0.05).

Results

At 0 and 24 hours, 1/T1 and τc data revealed significant differences between GVII and both the non‐irradiated (GI, GIII), and irradiated (GII, GIV) groups. At 48 hours, only the difference in τc between GVII and the irradiated group (GVI) remained significant.

Conclusions

Spin‐lattice data reflected significant changes in tissues induced by the burn and a tendency towards control values for all burned groups. Meanwhile, the τc value of GVI suggests the possibility of enhanced reparative effects attributable to chaotic intra‐ and inter‐molecular energy transport to biopolymers in injured soft‐tissue. Lasers Surg. Med. 33:190–198, 2003. © 2003 Wiley‐Liss, Inc.