bluuebunny/crossref_metadata_2025 huggingface dataset:
Pathogenicity by Parenteral Injection of Fowl Adenovirus Isolated from Gizzard Erosion and Resistance to Reinfection in Adenoviral Gizzard Erosion in Chickens
Authors: Ono M. , Okuda Y. , Shibata I. , Sato S. , Okada K.
Published: September 2004

The pathogenicity of a serotype-1 fowl adenovirus (FAV-99ZH), which causes adenoviral gizzard erosion by oral inoculation in chickens, was investigated in specific pathogen-free white leghorn chickens. In trial 1, 14 chickens were inoculated intravenously with the virus at 21 days of age and euthanatized for necropsy within 1–14 days of inoculation. Gizzard erosion was grossly observed from day 7 postinoculation (PI), and histologically, FAV-99ZH antigen-positive, basophilic intranuclear inclusion bodies were seen in the gizzard lesions from day 7 to 11 PI. Necrotizing pancreatitis, and cholecystitis and cholangitis associated with the inclusions were observed from day 3 to 14 PI (pancreatitis) and from day 5 to 9 PI (cholecystitis and cholangitis), respectively. The inclusions were also observed in the epithelial cells of the cecal tonsils from day 3 to 5 PI. The virus was recovered from samples of the lesions. It was revealed that FAV-99ZH causes not only gizzard erosion but also pancreatitis, cholecystitis, and cholangitis by intravenous inoculation in chickens. In trial 2, 10 chickens were inoculated orally with the virus twice, at 13 and 36 days of age, and euthanatized for necropsy within 4–17 days after reinfection. Macroscopically, focal gizzard lesions were observed; however, neither necrosis nor inclusions were observed by microscopy. Moreover, FAV was not recovered from the gizzard or rectum of any of the chickens at necropsy. This suggests that the gizzard lesions occurred as a result of the primary infection, and that the chickens were able to resist reinfection.
Indicators of Malignancy of Canine Adrenocortical Tumors: Histopathology and Proliferation Index
Authors: Labelle P. , Kyles A. E. , Farver T. B. , de Cock H. E. V.
Published: September 2004

Tumors of the adrenal cortex account for 10–20% of the naturally occurring Cushing's syndrome diagnosed in dogs. Differentiating between adrenocortical adenoma and carcinomas is often difficult. The purposes of this study were to determine which histopathologic criteria can be used as markers for malignancy in canine adrenocortical tumors and the relevance of the proliferation marker, Ki-67, for differentiation between cortical adenomas and carcinomas. Twenty-six adrenocortical carcinomas, 23 adenomas, and 11 normal adrenal glands were examined. Morphologic criteria significantly associated with adrenocortical carcinomas included a size larger than 2 cm in diameter, peripheral fibrosis, capsular invasion, trabecular growth pattern, hemorrhage, necrosis, and single-cell necrosis, whereas hematopoiesis, fibrin thombi, and cytoplasmic vacuolation were significantly associated with adrenocortical adenomas. The mean (± SD) proliferation index, measured by immunohistochemistry for the Ki-67 antigen, was 9.3 ± 6.3% in carcinomas, 0.76 ± 0.83% in adenomas, and 0.58 ± 0.57% in normal adrenal glands. The Ki-67 proliferation index was significantly higher in carcinomas compared with adenomas and normal adrenal glands. A threshold value of the proliferation index of 2.4% reliably separated carcinomas from adenomas. Based on these results, it appears that thorough evaluation of morphologic features combined with immunohistochemical assessment of the proliferation index is extremely useful for differentiating between adrenocortical adenomas and carcinomas in dogs.
Kinetics of Infection and Effects on the Placenta of Clamydophila abortus in Experimentally Infected Pregnant Ewes
Authors: Navarro J. A. , García de la Fuente J. N. , Sánchez J. , Martínez C. M. , Buendía A. J. , Gutiérrez-Martín C. B. , Rodriguez-Ferri E. F. , Ortega N. , Salinas J.
Published: September 2004

A Chlamydophila abortus-induced abortion model was carried out on the basis of the experimental infection of ewes at day 75 of gestation. The infection induced abortions and the birth of weak lambs during the last 3 weeks of pregnancy. To study the kinetics of the infection in the placenta and in other organs, infected ewes were killed at 105, 120, and 130 days of gestation and also several days after abortion or parturition. Infected ewes developed a systemic infection that caused a mild and transient pneumonia and focal hepatitis. Pathologic changes were observed in placentas at 120 day of gestation, although the lesions varied between animals and even between placentomes of the same placenta. The first placental area infected was the maternal stroma and epithelium next to the intercaruncular areas, where neutrophilic response seemed to control the infection. A substantial degree of multiplication of C. abortus was then observed in the trophoblast cells of the placentome, periplacentomal choriallantoic membranes, and hilius, with an inflammatory exudate composed mainly of neutrophils, some macrophages, and very scarce lymphocytes. After abortion, the lesions affected the intercotyledonary areas of the aborted placentas, whereas in the uterus significant lymphocyte infiltration was observed, together with a rapid decrease of the C. abortus antigen in the degenerated caruncular tissues.
Disseminated Infection with Balamuthia mandrillaris in a Dog
Authors: Foreman O. , Sykes J. , Ball L. , Yang N. , De Cock H.
Published: September 2004

Bilateral chronic granulomatous nephritis and meningoencephalitis were diagnosed on necropsy of a 2-year-old male Great Dane dog. The causative agent was identified as Balamuthia mandrillaris, based on morphologic features, immunohistochemical staining, and deoxyribonucleic acid detection using the polymerase chain reaction with newly designed primer pairs. Trophozoite and cystic forms of the amoeba were evident within the kidneys and brain parenchyma. This is the first report on a B. mandrillaris infection in a dog.
Intracranial Fusariosis: A Novel Cause of Fungal Meningoencephalitis in a Dog
Authors: Evans J. , Levesque D. , de Lahunta A. , Jensen H. E.
Published: September 2004

The Fusarium species are a group of saprophytic fungal organisms that are occasionally the cause of opportunistic infections in humans and animals. Central nervous system disease associated with a Fusarium species is most commonly described in horse, resulting in equine leukoencephalomalacia. This report describes a 2-year-old, spayed, female German Shepherd Dog with meningoencephalitis secondary to infection with Fusarium spp. Meningoencephalitis in dogs secondary to a species of Fusarium has not been previously reported. The diagnosis was made based on the histopathologic examination of brain tissues postmortem and special immunohistochemical stains specific for Fusarium solani. The clinical signs in this dog were indicative of multifocal brain disease and included seizures and a paradoxical vestibular syndrome. The clinical findings, diagnostic and histopathologic test results, and the comparative characterizations of other disseminated fungal diseases, especially aspergillosis, are described.
Type A–like Retroviral Particles in a Metastatic Intestinal Adenocarcinoma in an Emerald Tree Boa (Corallus caninus)
Authors: Orós J. , Lorenzo H. , Andrada M. , Recuero J.
Published: September 2004

A metastatic intestinal papillary adenocarcinoma was diagnosed histologically in an emerald tree boa ( Corallus caninus). Metastasis was detected in the liver, both kidneys, lung, and coelomic wall. Ultrastructural examination of the metastatic intestinal epithelial cells in the liver revealed the presence of a moderate number of viral particles that most closely resembled type A retroviral particles and were mainly associated with granular endoplasmic reticulum membranes. This case is the first description of type A-like retroviral particles in a neoplasm of a snake. The role of the virions in the etiology of the intestinal adenocarcinoma is uncertain. In addition, this is the first confirmed report of a metastatic intestinal adenocarcinoma in a snake.
Congenital Fetal Rhabdomyoma in a Foal
Authors: Meyerholz D. K. , Caston S. S. , Haynes J. S.
Published: September 2004

An Appaloosa filly was born with a ventral midline, approximately 8 × 12 × 15 cm subcutaneous cervical mass. The nonencapsulated mass was composed of interlacing and haphazard bundles of spindle cells on moderate to abundant loose myxomatous stroma. A moderate number of cells showed cross striations with minor nuclear variation and a low mitotic rate. Immunohistochemical staining for myoglobin, desmin, actin, vimentin, and S-100 was positive and negative for glial fibrillar antigen and keratin. Rhabdomyomas are rare benign tumors of striated muscle. Rhabdomyomas described previously in the veterinary literature are analogous to the “adult form” of human rhabdomyoma. This is the first report of a veterinary case that 1) clinically and histologically parallels the “fetal form” in human rhabdomyoma and 2) describes a congenital extracardiac rhabdomyoma.
Pulmonary Neoplasia in Two Llamas (Lama glama)
Authors: Ramos-Vara J. A. , Loiacono C. M. , Williams F. , Pardo I. , Lakritz J.
Published: September 2004

Two llamas with pulmonary tumors were examined. Llama No. 1 had multiple nodules throughout the lung that consisted histologically of solid clusters of polygonal to spindle cells with rare glandular differentiation. Intravascular emboli were common. Similar neoplastic masses were present in the kidney, heart, and liver. Immunohistochemically, neoplastic cells were positive for broad-spectrum cytokeratins (CKs), high- molecular weight CKs, CKs 5/6, and vimentin. The diagnosis was pulmonary carcinoma. Llama No. 2 had pulmonary nodules without extrapulmonary involvement. Microscopically, neoplastic cells formed acini lined by simple epithelium and solid cords of squamous cells that sometimes surrounded acini. Neoplastic cells were strongly positive for broad-spectrum CKs and weakly positive for thyroid transcription factor-1. The diagnosis was adenosquamous carcinoma. Pulmonary tumors account for 23% of neoplasms in South American camelids in our laboratory, making this the second most common type of neoplasm after lymphosarcoma.
Oligodendroglioma in the Cervical Spinal Cord of a Dog
Authors: Mamom T. , Meyer-Lindenberg A. , Hewicker-Trautwein M. , Baumgärtner W.
Published: September 2004

A 7-year-old male castrated Yorkshire Terrier dog developed slowly progressive neurologic disturbances consisting of difficulties in moving the neck, lack of proprioception, and tetraparesis 4 months prior its death. Neurologic examination, computer tomography, and myelography resulted in the tentative diagnosis of intramedullary cervicothoracic spinal cord lesion. At necropsy, an intramedullary cervical spinal cord mass between C5 and C6 was noticed. Histologically, cells of this well-demarcated, nonencapsulated neoplasm were arranged in sheaths or cords separated by a fine fibrovascular stroma. The polygonal to round tumor cells were characterized by moderate pale, basophilic, and vacuolar cytoplasm and round to slightly oval, centrally located nuclei with fine-stippled heterochromatin, a single nucleolus, and a very low mitotic activity. Tumor cells lacked glial fibrillary acidic protein, vimentin, factor VIII-related, and cytokeratin antigen expression. Histologic and immunohistochemical findings led to the diagnosis of a cervical spinal cord oligodendroglioma.
Leukoencephalitis Associated with Selective Viral Replication in the Brain of a Pony with Experimental Chronic Equine Infectious Anemia Virus Infection
Authors: Oaks J. L. , Long M. T. , Baszler T. V.
Published: September 2004

Neurologic disease occurs sporadically in horses infected with the equine infectious anemia virus (EIAV). This report describes a case of clinically severe neurologic disease in a pony experimentally infected with EIAV. This pony did not have fever or anemia, which are the characteristic clinical signs of disease. The histopathologic changes were characterized as lymphohistiocytic periventricular leukoencephalitis. Polymerase chain reaction and in situ hybridization data showed that the brain lesions were directly associated with viral replication and that high-level viral replication occurred selectively within the lesion and not in other tissues. These findings suggest that EIAV-associated neurologic disease is the direct result of viral replication.
Multifocal Pleural Cystic Squamous Metaplasia in a Horse with Chronic Obstructive Bronchopneumonia
Authors: Baum B. , Hewicker-Trautwein M. , Wohlsein P.
Published: September 2004

A 10-year-old Knabstrupper stallion was euthanatized because of severe dyspnea and exercise intolerance. Postmortem examination revealed diffuse severe alveolar emphysema and chronic fibrosing pleuritis of the caudal lung. Parts of both caudal lung lobes were covered with multiple raised firm gray to yellow plaques. Histologically, these areas consisted of circumscribed pleural fibroses and cysts of metaplastic keratinizing squamous epithelium. Immunohistochemistry revealed intense labeling for cytokeratins 5/6 and 10. In addition, caudal lung lobes were severely affected by a chronic partially obliterative bronchiolitis and peribronchiolitis with multifocal pleural involvement.
Operational Restructuring Charges and Post‐Restructuring Performance*
Authors: ATIASE ROWLAND K. , PLATT DAVID E. , TSE SENYO Y.
Published: September 2004

Firms incur restructuring charges as a result of actions intended to improve their operating performance. However, there is little evidence on whether restructuring charges are associated with improved performance. We examine a sample of firms reporting restructuring in 1991‐93 and find that the restructuring firms' earnings increase over the levels immediately before restructuring. Compared with a control sample of firms that report no restructuring, the restructuring firms improve their earnings and operating income, but evidence for improvements in cash flow from operations is mixed. In regression analysis, we find that restructuring charges are significantly positively associated with post‐restructuring changes in earnings relative to the restructuring year, but this association is largely driven by firms with multiple restructurings and firms reporting losses in the restructuring year. We find no association between restructuring charges and post‐restructuring changes in earnings relative to the year before restructuring. Restructuring charges are significantly positively associated with post‐restructuring changes in operating income and cash flow from operations for firms with multiple restructurings. In summary, restructuring charges are associated with improved earnings, but our results suggest that restructuring in the early 1990s did not necessarily guarantee improved operating performance.
Conserved Repressive Regulation of Connective Tissue Growth Factor/Hypertrophic Chondrocyte-Specific Gene 24 (ctgf/hcs24) Enabled by Different Elements and Factors among Vertebrate Species
Authors: Mukudai Y. , Kubota S. , Takigawa M.
Published: September 2004

CTGF/Hcs24 is a multifunctional growth factor that potentiates the growth and differentiation of various cells.
Our previous study revealed that the 3'-UTR of mammalian CTGF/Hcs24 mRNA contains a small segment
that represses the gene expression in cis fashion. In
this study, we isolated and characterized a chicken
CTGF/Hcs24 cDNA clone. Chicken ctgf/hcs24 mRNA
showed highly conserved homology in the ORF to that
of mammalian species, whereas the homology in the
3'-UTR was relatively low. Northern blotting analysis
revealed that chicken ctgf/hcs24 mRNA was expressed most strongly in cartilage, and also in brain,
lung, heart, but faintly in liver. Thereafter we analyzed
the functional potential of the 3'-UTR of ctgf/hcs24
cDNA to regulate its gene expression by reporter gene
assay, and found that it repressed gene expression in
cis fashion, specifically in avian cells, but not in mammalian cells. Conversely, the mammalian 3'-UTR
showed less repressive activity in avian cells than in
mammalian cells. Deletion analysis showed that a segment near the polyadenyl tail of the 3'-UTR of chicken
ctgf/hcs24 played an important functional role, unlike
in the mammalian species. Thus, we uncovered a novel
mode of functional conservation of the ctgf/hcs24 3
UTR among vertebrate species mediated by different
factors.
Different Sensitivities of Mutants and Chimeric Forms of Human Muscle and Liver Fructose- 1,6-Bisphosphatases towards AMP
Authors: Rakus D. , Tillmann H. , Wysocki R. , Ulaszewski S. , Eschrich K. , Dzugaj A.
Published: September 2004

AMP is an allosteric inhibitor of human muscle and liver fructose-1,6-bisphosphatase (FBPase). Despite strong similarity of the nucleotide binding domains, the muscle enzyme is inhibited by AMP approximately 35 times stronger than liver FBPase: I0.5 for muscle and for liver FBPase are 0.14 uM and 4.8 uM, respectively. Chimeric human muscle (L50M288) and chimeric human liver enzymes (M50L288), in which the N-terminal residues (1-50) were derived from the human liver and human muscle FBPases, respectively, were inhibited by AMP 2-3 times stronger than the wild-type liver enzyme. An amino acid exchange within the Nterminal region of the muscle enzyme towards liver FBPase (Lys20→Glu) resulted in 13-fold increased I0.5 values compared to the wild-type muscle enzyme. However, the opposite exchanges in the liver enzyme (Glu20→Lys and double mutation Glu19→Asp/Glu20→Lys) did not change the sensitivity for AMP inhibition of the liver mutant (I0.5 value of 4.9 uM). The decrease of sensitivity for AMP of the muscle mutant Lys20→Glu, as well as the lack of changes in the inhibition by AMP of liver mutants Glu20→Lys and Glu19→Asp/Glu20→Lys, suggest a different mechanism of AMP binding to the muscle and liver enzyme.
Identification by Phage Display of the Linear Continuous MRPr1 Epitope in the Multidrug Resistance-Associated Protein (MRP1)
Authors: Flego M. , Mennella V. , Moretti F. , Poloni F. , Dupuis M.L. , Ascione A. , Barca S. , Felici F. , Cianfriglia M.
Published: September 2004

In order to study the structure of the multidrug resistance-associated protein (MRP1), which is one of the
most important members of the ATP-binding cassette
(ABC) protein family acting as drug-efflux systems, we
have developed an epitope mapping-based strategy.
By means of the mAb MRPr1, we have immunoselected clones from two distinct random peptide libraries
displayed on phages and have identified several peptide sequences mimicking the internal conformation
of this 190 kDa multidrug transporter protein. Phage
clones able to block the immunolabeling of the MRPr1
antibody to MRP1-overexpressing multidrug resistance (MDR) H69/AR cells were isolated and, after sequencing the corresponding inserts, their amino acid
sequence was compared to that of MRP1. This analysis led to the identification of the consensus sequence
L.SLNWED, corresponding to the MRP1 segment
LWSLNKED (residues 241–248). This MRP1sequence
is partially overlapping with the MRPr1 epitope GSDL
WSLNKE (residues 238–247) previously mapped using peptide scanning techniques. These results
demonstrate the high reliability of phage display technology to study not only the topography of complex
integral membrane proteins such as MRP1, but also to
help identify critical residues participating in the formation of the epitope structure.
The Cellular Control Enzyme PolyADP Ribosyl Transferase Is Eliminated in Cultured Fanconi Anemia Fibroblasts at Confluency
Authors: Ramirez M.H. , Adelfalk C. , Kontou M. , Hirsch-Kauffmann M. , Schweiger M.
Published: September 2004

Fanconi anemia (FA) is a hereditary disease of unknown pathogenic mechanisms, although mutations in seven different genes can be causative. Six of these genes have been cloned and sequenced. Only slight homology to the DNA of any other known gene has been found with the exception of FANCG which is identical to XRCC9. The function of these genes, including XRCC9, is presently unknown. Since pADP ribosyl transferase (pADPRT) plays a role in apoptosis, and apoptosis is affected in FA cells, we studied the correlation between pADPRT and FA cells. We reinvestigated the previously reported lack of pADPRT activity in fibroblasts from patients with Fanconi anemia. Here we describe the role of the lower redox potential of FA cells and demonstrate that this is an efficient strategy in the prevention of cell death due to the lack of energy under oxidative stress. This strategy is advantageous for the cells under the nonreplicative condition of confluency in which the risk of mutation is low and the prevention of apoptosis permits cell survival. pADPRT is not diminished to the same extent in all complementation groups of FA. It is prominent in FANCA, FANCG and FANCF cells, indicating that these genes control pADPRT diminution. Our experiments suggest that the pADPRT level is linked with the oxidoreduction reactions seen in FA.
‘Piggy-Back’ Transport of Xenopus Hyaluronan Synthase (XHAS1) via the Secretory Pathway to the Plasma Membrane
Authors: Müllegger J. , Rustom A. , Kreil G. , Gerdes H.-H. , Lepperdinger G.
Published: September 2004

Hyaluronan is the sole glycosaminoglycan whose biosynthesis takes place directly at the plasma membrane. The mechanism by which hyaluronan synthase (HAS) becomes inserted there, as well as the question of how the enzyme discriminates between particular membrane species in polarized cells, are largely unknown. In vitro translation of HAS suggested that the nascent protein becomes stabilized in the presence of microsomal membranes, but would not insert spontaneously into membranes after being translated in the absence of those. We therefore monitored the membrane attachment of enzymatically active fusion proteins consisting of Xenopus HAS1 and green fluorescent protein shortly after de novo synthesis in Vero cells. Our data strongly suggest that HAS proteins are directly translated on the ER membrane without exhibiting an N-terminal signal sequence. From there the inactive protein is transferred to the plasma membrane via the secretory pathway. For unknown reasons, HAS inserted into membranes other than the plasma membrane remains inactive.
A Novel Influenza A Virus Activating Enzyme from Porcine Lung: Purification and Characterization
Authors: Sato M. , Yoshida S. , Iida K. , Tomozawa T. , Kido H. , Yamashita M.
Published: September 2004

Proteolytic activation of hemagglutinin, an envelope glycoprotein of the influenza virus, by host proteases is essential for infection and proliferation of the virus. However, there is no well-defined, inherent source of host proteases in man or swine, both of which are natural hosts for human influenza viruses. We have recently isolated a 32 kDa protein in a high salt extract from porcine lungs, which possess the hemagglutinin processing activity. In this study, we attempted to purify another hemagglutinin processing enzyme from porcine lung. The purified enzyme, named tryptase TC30, exhibited a molecular mass of about 30 kDa by SDS-PAGE and 28.5 kDa by gel filtration chromatography, suggesting that it is a monomer. Tryptase TC30 cleaved peptide substrates with Arg at the P1 position, and preferentially substrates with the Ser-Ile-Gln-Ser-Arg sequence corresponding to the HA cleavage site sequence of the A/PR/8/34 influenza virus. Among various inhibitors tested, trypsintype serine protease inhibitors, such as aprotinin, antipain, benzamidine and leupeptin, efficiently inhibited the proteolytic activity of the enzyme. The N-terminal 40 amino acid sequence of tryptase TC30 exhibits more than 60% homology to mast cell tryptases from mice MCP-6 and human tryptase-α and -β. These data indicate that tryptase TC30, the 30 kDa enzyme from porcine lung, is a novel hemagglutinin-cleaving enzyme.
A Homodimeric Sporamin-Type Trypsin Inhibitor with Antiproliferative, HIV Reverse Transcriptase-Inhibitory and Antifungal Activities from Wampee (Clausena lansium) Seeds
Authors: Ng T.B. , Lam S.K. , Fong W.P.
Published: September 2004

A homodimeric trypsin inhibitor with a molecular
mass of 54 kDa was isolated from the seeds of
Clausena lansium (Lour) Skeels with a very simple
procedure comprising extraction with an aqueous
buffer and ion exchange chromatography on CM-cellulose.
It inhibited trypsin with an IC50 of 2.2 nM but was
without any inhibitory effect on chymotrypsin and proteinase
K. The uptake of MTT by human leukemia
HL60 and hepatoma Hep G2 cells was inhibited with
an IC50 of 100 uM. Translation in the cellfree rabbit
reticulocyte lysate system was inhibited with an IC50
of 3.6 uM. The activity of HIV-1 reverse transcriptase
was reduced in the presence of the trypsin inhibitor.
The trypsin inhibitor exerted antifungal activity toward
Physalospora piricola but not Mycosphaerella
arachidicola, Botrytis cinerea, Fusarium oxysporum
or Coprinus comatus.
A Bioinformatic Approach to the Identification of Candidate Genes for the Development of New Cancer Diagnostics
Authors: Musumarra G. , Barresi V. , Condorelli D.F. , Scirè S.
Published: September 2004

A multivariate analysis of the National Cancer Institute
gene expression database is reported here. The
soft independent modelling of a class analogy approach
achieved cell line classification according to
histological origin. With the PCA method, based on
the expression of 9605 genes and ESTs, classification
of colon, leukaemia, renal, melanoma and CNS cells
could be performed, but not of lung, breast and ovarian
cells. Another multivariate procedure, called partial
least squares discriminant analysis (PLS-DA),
provides bioinformatic clues for the selection of a limited
number of gene transcripts most effective in discriminating
different tumoral histotypes. Among
them it is possible to identify candidates in the development
of new diagnostic tests for cancer detection
and unknown genes deserving high priority in further
studies. In particular, melan-A, acid phosphatase 5,
dopachrome tautomerase, S100-β and acid ceramidase
were found to be among the most important
genes for melanoma. The potential of the present
bioinformatic approach is exemplified by its ability to
identify differentiation and diagnostic markers already
in use in clinical settings, such as protein S-100,
a prognostic parameter in patients with metastatic
melanoma and a screening marker for melanoma
metastasis.
Recombinant RNase Z Does Not Recognize CCA as Part of the tRNA and Its Cleavage Efficieny Is Influenced by Acceptor Stem Length
Authors: Schiffer S. , Rösch S. , Marchfelder A.
Published: September 2004

One of the essential maturation steps to yield functional tRNA molecules is the removal of 3'-trailer sequences by RNase Z. After RNase Z cleavage the tRNA nucleotidyl transferase adds the CCA sequence to the tRNA 3terminus, thereby generating the mature tRNA. Here we investigated whether a terminal CCA triplet as 3'-trailer or embedded in a longer 3'- trailer influences cleavage site selection by RNase Z using three activities: a recombinant plant RNase Z, a recombinant archaeal RNase Z and an RNase Z active wheat extract. A trailer of only the CCA trinucleotide is left intact by the wheat extract RNase Z but is removed by the recombinant plant and archaeal enzymes. Thus the CCA triplet is not recognized by the RNase Z enzyme itself, but rather requires cofactors still present in the extract. In addition, we investigated the influence of acceptor stem length on cleavage by RNase Z using variants of wildtype tRNATyr. While the wild type and the variant with 8 base pairs in the acceptor stem were processed efficiently by all three activities, variants with shorter and longer acceptor stems were poor substrates or were not cleaved at all.
Retinoids Inhibit Human Epidermal Keratinocyte RNase P Activity
Authors: Papadimou E. , Pavlidou D. , Séraphin B. , Tsambaos D. , Drainas D.
Published: September 2004

Ribonuclease P (RNase P) is a ubiquitous and essential enzyme that endonucleolytically cleaves all tRNA precursors to produce the mature 5'-end. We have investigated the effect of synthetic rertinoids (all-trans retinoic acid, acitretin) and arotinoids (Ro 13-7410, Ro 15-0778, Ro, 13-6298 and Ro 15-1570) on RNase P activity isolated for the first time from normal human epidermal keratinocytes (NHEK). All tested compounds but one (Ro 15-1570) revealed a dose-dependent inhibition of RNase P activity, indicating that they may have a direct effect on tRNA biogenesis. Detailed kinetic analysis showed that all retinoids behave as classic competitive inhibitors. On the basis of the Ki values Ro 13-7410 was found to be the strongest inhibitor among all compounds tested.
Synthesis and Characterization of EMPO-Derived 5,5-Disubstituted 1-Pyrroline N-Oxides as Spin Traps Forming Exceptionally Stable Superoxide Spin Adducts
Authors: Stolze K. , Udilova N. , Rosenau T. , Hofinger A. , Nohl H.
Published: September 2004

EMPO [5-(ethoxycarbonyl)-5-methyl-1-pyrroline N-oxide]
is a highly hydrophilic cyclic nitrone spin trap,
whose superoxide adduct is considerably more stable
(t1/2=8.6 min) than DMPO (5,5-dimethyl-1-pyrroline
Noxide, t1/2=45 s). EPR spectra of spin adducts of
EMPO and its derivatives are very similar to those of
the respective DMPO spin adducts, in contrast to the
rather complex spectra obtained using DEPMPO [5-
(diethoxyphosphoryl)-5-methyl-1-pyrroline Noxide].
Several EMPO derivatives, with both the ethoxycarbonyl
group and the methyl group at position 5 of the
pyrroline ring being replaced by other substituents,
were synthesized and characterized by 1H and 13C
NMR spectroscopy. Thus, a series of derivatives was
obtained that exhibit large differences in the stability
of their superoxide adducts, ranging from less than
one to more than 25 min. The stability of the superoxide
adducts was mainly determined by the steric environment
of the nitroxyl group: in compounds with
less bulky 5-alkoxycarbonyl substituents the nitroxyl
group is sterically less shielded, which resulted in a
lower stability of the superoxide adducts. The spin
density distribution, as obtained from DFT computations,
was found to be nearly identical for all compounds,
so that in contrast to the steric influences the
spin density did not seem to be a crucial factor for the
stability of the superoxide adducts.
Modulation of the Chymotrypsin-Like Activity of the 20S Proteasome by Intracellular Redox Status: Effects of Glutathione Peroxidase-1 Overexpression and Antioxidant Drugs
Authors: Kretz-Remy C. , Arrigo A.-P.
Published: September 2004

ATP- and ubiquitin-independent proteolysis by the 20S proteasome is responsible for the selective degradation of oxidized proteins. In vitro, the 20S proteasome shows an increased proteolytic activity toward oxidized polypeptides and the suc-LLVY-MCA peptide specific for its chymotrypsinlike activity. We have analyzed the effect of the intracellular redox status on the chymotrypsin-like activity of the 20S proteasome in human T47D cells overexpressing the detoxifiant enzyme seleno-glutathione peroxidase-1 (GPx-1). We report a 30% decreased activity of the chymotrypsin-like activity in cells overexpressing GPx-1. This phenomenon correlated with a 2-fold increase in IκBα half-life, a protein whose basal turnover is 20S proteasome-dependent. Following exposure to H2O2, these cells showed a seleno-dependently decreased accumulation of intracellular reactive oxygen species and 20S proteasome chymotrypsin like activity. Similar results were obtained in HeLa cells transiently overexpressing human GPx-1. Moreover, exposure of HeLa cells to antioxidant compounds reduced the proteasome 20S chymotrypsin like activity. In contrast, no effects were observed when HeLa cell extracts used to determine proteasome activity were incubated with either reduced or oxidized glutathione. These results suggest that GPx-1 activity or proreducing conditions can downregulate basal 20S proteasome activity. Hence, the intracellular redox status, probably through the level of oxidized proteins, is an important element that can either activate or down-regulate the 20S proteasome chymotrypsin-like activity in living cells.
Testis-Specific Expression of the Nuclear Form of Phospholipid Hydroperoxide Glutathione Peroxidase (PHGPx)
Authors: Moreno S. G. , Laux G. , Brielmeier M. , Bornkamm G. W. , Conrad M.
Published: September 2004

The selenoprotein phospholipid hydroperoxide glutathione
peroxidase (PHGPx) is present in at least
three different isoforms in testis: as a cytosolic, as a
mitochondrial, and as a nuclear protein. We have recently
shown that a sperm nucleus-specific glutathione
peroxidase (snGPx) is identical to the mitochondrial
and cytosolic forms of PHGPx apart from
its N-terminus. This arginine-rich N-terminus of snGPx, reminiscent of protamines, is encoded by an alternative
exon located in the first intron of the PHGPx gene and is responsible for nuclear localisation and chromatin binding of snGPx [Pfeifer et al., FASEB J.
15 (2001), pp. 1236-1238]. By using a combination of
techniques including selective cloning of mRNA 5'
ends, RT-PCR, and S1 analyses, we provide evidence
that the transcript encoding the nuclear form is generated
by transcription initiation at an alternative promoter
and not by alternative splicing. We show that
the major transcription start region is located at 12
to 14 upstream of the AUG translation initiation site
of the sperm nucleus-specific exon and lacks a TATA
box. Two minor TATA-less transcription initiation sites
are located at around -30 and -45. We have shown by
in situ hybridisation that snGPx expression in testis,
like protamine expression, is restricted to late stages
of spermatogenesis whereas PHGPx expression is
only found in spermatocytes and early spermatids.
These findings have to be taken into account when
studying either the differential regulation of PHGPx
and snGPx expression in testis or the impact of putative
mutations in snGPx on male fertility in man.
Regulation of Gene Transcription by a Constitutively Active Mutant of Activating Transcription Factor 2 (ATF2)
Authors: Steinmüller L. , Thiel G.
Published: September 2004

Activating transcription factor 2 (ATF2) belongs to the family ofbasic region leucinezipper (bZIP) proteins that are characterized by the presence of a basic domain that functions as the DNAbinding domain and a leucine zipper domain that is required for dimerization. Together with bZIP proteins of the Fos and Jun families, ATF2 constitutes the AP-1 transcription factor complex. The biological activity of ATF2 is controlled by phosphorylation of two threonine residues within the N-terminal activation domain. Unphosphorylated ATF2 is trancriptionally silent, excluding simple overexpression studies to identify transcriptional targets of ATF2. We therefore decided to construct a constitutively active ATF2 mutant that would allow us to uncouple the investigation of transcriptional targets and biological functions of ATF2 from the variety of signaling pathways that lead to an activation of ATF2. We exchanged the phosphorylationdependent activation domain of ATF2 with the constitutively active transcriptional activation domain of the transcription factor CREB2. In transient transfection experiments, this constitutively active ATF2 mutant stimulated c-jun, tumor necrosis factor α, and Fas ligand promoter activities. The transcriptional activity of the constitutively active ATF2 mutant could be impaired by dominantnegative forms of ATF2 or c-jun, indicating that ATF2 and c-jun utilize a similar dimerization code. In contrast, a dominantnegative CREB2 mutant did not impair ATF2-mediated transcriptional activation, suggesting that CREB2 exhibits a different dimerization specificity than ATF2 or c-jun.
Solvent Isotope Effect on the Reaction Catalysed by the Pyruvate Dehydrogenase Complex from Escherichia coli
Authors: Liu X. , Bisswanger H.
Published: September 2004

The pyruvate dehydrogenase from Escherichia coli showed a primary kinetic isotope effect when its overall reaction or the partial reaction of the pyruvate dehydrogenase component were tested in deuterium oxide. The Michaelis constants for pyruvate were nearly unchanged, but the maximum velocities in water and deuterium oxide differed, their ratio being DV = 1.7 for the overall reaction and DV = 2.1 for the E1p reaction. The pH profile and, accordingly, the δpK1 and δpK2 values were shifted by 0.6 units to higher pL values. A linear proton inventory curve was obtained when varying the atom fractions of protons relative to deuterons from 100 to 0%. This is an indication for a single proton transfer. It is proposed that this relatively weak primary isotope effect may be caused by the protonation of the N1 nitrogen at the pyrimidine ring of the cofactor by an adjacent glutamate residue. The proton of its carboxylic group exchanges very fast with deuterons of the solvent.
The Circadian Clock of the Unicellular Eukaryotic Model Organism Chlamydomonas reinhardtii
Authors: Mittag M. , Wagner V.
Published: September 2004

The green unicellular alga Chlamydomonas reinhardtii, also called 'green yeast', emerged in the past years as a model organism for specific scientific questions such as chloroplast biogenesis and function, the composition of the flagella including its basal apparatus, or the mechanism of the circadian clock. Sequencing of its chloroplast and mitochondrial genomes have already been completed and a first draft of its nuclear genome has also been released recently. In C. reinhardtii several circadian rhythms are physiologically well characterized, and one of them has even been shown to operate in outer space. Circadian expression patterns of nuclear and plastid genes have been studied. The mode of regulation of these genes occurs at the transcriptional level, although there is also evidence for posttranscriptional control. A clock-controlled, phylogenetically conserved RNA-binding protein was characterized in this alga, which interacts with several mRNAs that all contain a common cis-acting motif. Its function within the circadian system is currently under investigation. This review summarizes the current state of the knowledge about the circadian system in C. reinhardtii and points out its potential for future studies.
Identification of Rhythmic Subsystems in the Circadian Cycle of Crassulacean Acid Metabolism under Thermoperiodic Perturbations
Authors: Bohn A. , Hinderlich S. , Hütt M.-T. , Kaiser F. , Lüttge U.
Published: September 2004

Leaves of the Crassulacean acid metabolism (CAM) plant Kalanchoë daigremontiana Hamet et Perrier de la Bâthie show overt circadian rhythms in net CO2 uptake, leaf conductance to water and intercellular CO2 concentration, which are entrained by periodic temperature cycles. To probe their sensitivity to thermoperiodic perturbations, intact leaves were exposed to continuous light intensity and temperature cycles with a period of 16 h, applying a set of different baseline temperatures and thermodriver amplitudes. All three overt rhythms were analyzed with respect to their frequency spectra and their phase relations with the thermodriver. For most stimulation protocols, stomatal conductance and net CO2 change were fully or partially entrained by the temperature pulses, while the internal CO2 concentration remained dominated by oscillations in the circadian range. Prolonged time series recorded for up to 22 d in continuous light underline the robustness of these circadian oscillations. This suggests that the overt circadian rhythm of net CO2 uptake in CAM results from the interaction of two coupled original systems: (i) an endogenous cycle of CO2 fixation in the mesophyll, showing very robust periodic activity, and (ii) stomatal movements that respond to environmental stimuli independently of rhythmic processes in the mesophyll, and thus modulate the gas exchange amplitude.
Age-Dependent Effects of Conditioning on Cholinergic and Vasopressin Systems in the Rat Suprachiasmatic Nucleus
Authors: Biemans B. A. M. , Zee E. A. Van der , Daan S.
Published: September 2004

Active shock avoidance was used to explore the impact
of behavioural stimulation on the neurochemistry
of the suprachiasmatic nucleus. We have found
previously that the expression of muscarinic acetylcholine
receptors in the suprachiasmatic nucleus of
young rats was significantly enhanced 24 hours after
fear conditioning. Here, we investigated whether this
observation is age-dependent. We used 26 month-old
Wistar rats with a deteriorated circadian system, and
compared them with young rats (4 months of age)
with an intact circadian system. Vasopressin, representing
a major output system of the suprachiasmatic
nucleus, was studied in addition to muscarinic receptors.
Young rats showed a significant increase in
immunostaining for muscarinic acetylcholine receptors
24 h after training, corroborating earlier observations.
Aged rats did not show such an increase. In
contrast, aged rats did show an increase in vasopressin
immunoreactivity 24 h after fear conditioning,
both at the level of content and cell number, while
young rats did not reveal a significant rise. Thus, it
seems that these two neurochemical systems in the
suprachiasmatic nucleus are regulated independently.
The results further demonstrate that the circadian
pacemaker is influenced by fear conditioning in an
age-dependent manner.
Caspases and Neuronal Development
Authors: Ryan C. A. , Salvesen G. S.
Published: September 2004

Recent developments have shown that inappropriate activation of apoptotic pathways contributes to many neurodegenerative diseases. The basic mechanisms that underlie apoptosis in neurodegenerative diseases are uncertain, although they likely represent the subversion of normal developmental programs. Several types of neuronal cell death have been reported, including autophagic and caspase-independent cell death. In this review we consider evidence for the participation of apoptotic caspases in neuronal development, and examine the hypothesis that differentiating neurons undergo stage-specific alterations in apoptosis sensitivity that may be due to caspase regulation. In addition, we present data supporting this hypothesis.
Procongopain from Trypanosoma congolense Is Processed at Basic pH: An Unusual Feature among Cathepsin L-Like Cysteine Proteases
Authors: Serveau C. , Boulangé A. , Lecaille F. , Gauthier F. , Authié E. , Lalmanach G.
Published: September 2004

Congopain, the major cysteine protease from Trypanosoma congolense, is synthesized as an inactive zymogen, and further converted into its active form after removal of the proregion, most probably via an autocatalytic mechanism. Processing of recombinant procongopain occurs via an apparent one-step or a multistep mechanism depending on the ionic strength. The auto-activation is pH-dependent, with an optimum at pH 4.0, and no activation observed at pH 6.0. After addition of dextran sulfate (10 ug/ml), an approx. 20-fold increase of processing (expressed as enzymatic activity) is observed. Furthermore, in the presence of dextran sulfate, procongopain can be processed at pH 8.0, an unusual feature among papainlike enzymes. Detection of procongopain and trypanosomal enzymatic activity in the plasma of T. congolenseinfected cattle, together with the capacity of procongopain to be activated at weakly basic pH, suggest that procongopain may be extracellularly processed in the presence of blood vessel glycosaminoglycans, supporting the hypothesis that congopain acts as a pathogenic factor in host-parasite relationships.
Heat Stress-Dependent DNA Binding of Arabidopsis Heat Shock Transcription Factor HSF1 to Heat Shock Gene Promoters in Arabidopsis Suspension Culture Cells in vivo
Authors: Zhang L. , Lohmann C. , Prändl R. , Schöffl F.
Published: September 2004

Using UV laser cross-linking and immunoprecipitation we measured the in vivo binding of Arabidopsis heat shock transcription factor HSF1 to the promoters of target genes, Hsp18.2 and Hsp70. The amplification of promoter sequences, co-precipitated with HSF1-specific antibodies, indicated that HSF1 is not bound in the absence of heat stress. Binding to promoter sequences of target genes is rapidly induced by heat stress, continues throughout the heat treatment, and declines during subsequent recovery at room temperature. The molecular mechanisms underlying the differences between Hsp18.2 and Hsp70 in the kinetics of HSF1/promoter binding and corresponding mRNA expression profiles are discussed.
Interaction of Maize Chromatin-Associated HMG Proteins with Mononucleosomes: Role of Core and Linker Histones
Authors: Lichota J. , Grasser K. D.
Published: September 2004

Two groups of plant chromatin-associated high mobility group (HMG) proteins, namely the HMGA family, typically containing four A/T-hook DNA-binding motifs, and the HMGB family, containing a single HMG-box DNA-binding domain, have been identified. We have examined the interaction of recombinant maize HMGA and five different HMGB proteins with mononucleosomes (containing approx. 165 bp of DNA) purified from micrococcal nuclease-digested maize chromatin. The HMGB proteins interacted with the nucleosomes independent of the presence of the linker histone H1, while the binding of HMGA in the presence of H1 differed from that observed in the absence of H1. HMGA and the HMGB proteins bound H1-containing nucleosome particles with similar affinity. The plant HMG proteins could also bind nucleosomes that were briefly treated with trypsin (removing the N-terminal domains of the core histones), suggesting that the histone N-termini are dispensable for HMG protein binding. In the presence of untreated nucleosomes and trypsinised nucleosomes, HMGB1 could be chemically crosslinked with a core histone, which indicates that the trypsin-resistant part of the histones within the nucleosome is the main interaction partner of HMGB1 rather than the histone N-termini. In conclusion, these results indicate that specific nucleosome binding of the plant HMGB proteins requires simultaneous DNA and histone contacts.
Physarum polycephalum Expresses a Dihydropteridine Reductase with Selectivity for Pterin Substrates with a 6-(1', 2-Dihydroxypropyl) Substitution
Authors: Wild C. , Golderer G. , Gröbner P. , Werner-Felmayer G. , Werner E. R.
Published: September 2004

Physarum polycephalum is one of few non-animal organisms capable of synthesizing tetrahydrobiopterin from GTP. Here we demonstrate developmentally regulated expression of quinoid dihydropteridine reductase (EC 1.6.99.7), an enzyme required for recycling 6,7-[8H]-dihydrobiopterin. Physarum also expresses phenylalanine-4-hydroxylase activity, an enzyme that depends on dihydropteridine reductase. The 24.4 kDa Physarum dihydropteridine reductase shares 43% amino acid identity with the human protein. A number of residues important for function of the mammalian enzyme are also conserved in the Physarum sequence. In comparison to sheep liver dihydropteridine reductase, purified recombinant Physarum dihydropteridine reductase prefers pterin substrates with a 6-(1', 2'-dihydroxypropyl) group. Our results demonstrate that Physarum synthesizes, utilizes and metabolizes tetrahydrobiopterin in a way hitherto thought to be restricted to the animal kingdom.