bluuebunny/crossref_metadata_2025 huggingface dataset:
Novel Molecular Photo-resists Based on the Cyclodextrin Derivatives Containing Fluorine Atoms and t-Butyl Ester Groups
Authors: Kudo Hiroto , Inoue Naomi , Nishimura Isao , Nishikubo Tadatomi
Published: April 2005

In this paper, we describe the syntheses, properties, and photo-induced deprotection of β-CD derivatives containing t-butyl ester groups and fluorine atoms. The β-CD derivative was obtained from the reaction of β-CD and t-butyl-α-(trifluoromethyl)acrylate. The obtained β-CD derivative had excellent solubility, good film-forming property, high thermal stability, and good transmittance with the VUV spectra. When the photo-chemical reaction of the β-CD derivative was performed with a photo acid generator in the film state upon UV irradiation, it was found that the photoinduced deprotection of t-butyl ester groups and decomposition of acetal moieties of gluconopyranose unit proceeded smoothly. Furthermore, we examined the patterning property of the β-CD derivative as the photo resist using triphenylsulfonium fluorosulfonate as a photo acid generator, and 2-heptanone as the solvent. From this result, it was observed that the resolution of a 100 nm line and space pattern could be obtained.
Identification of Subtypes of CYP2D Gene Rearrangements among Carriers of CYP2D6 Gene Deletion and Duplication
Authors: Ledesma María C , Agúndez José AG
Published: April 2005

Background: Cytochrome P450 2D6 (CYP2D6) is one of the best-known polymorphic drug-metabolizing enzymes. Rapidly evolving genotyping techniques permit the identification of single-nucleotide polymorphisms (SNPs) and thereby a prediction of individual metabolic capacities for CYP2D6 substrates. A considerable part of interindividual variability in CYP2D6 enzyme activity, however, is not related to SNPs but to gene deletions and duplications. Currently used genotyping methods assume that these gene rearrangements are homogeneous.

Methods: We analyzed the interindividual variability in CYP2D6 gene arrangements in genomic DNA from 740 Caucasian individuals by allele-specific PCR to identify common SNPs of the CYP2D6 gene that correspond to the variant alleles CYP2D6*3, *4, and *9. We investigated the presence and variability of CYP2D6*5 (gene deletion), CYP2D6x2 (gene duplication), and CYP2D6xn (gene amplification) by EcoRI and XbaI restriction fragment length polymorphism analyses and by long PCR plus KpnI and BamHI digestion. The presence of new mutations at the CYP2D locus was analyzed by sequencing.

Results: CYP2D6 gene rearrangements were present in >12% of individuals. Variability in the rearrangements regarding both gene deletion and gene duplication existed, and one of the unusual arrangements led to incorrect phenotype prediction. The frequency for carriers of unusual gene rearrangements was <0.3% (95% confidence interval, 0%–0.6%) in the population studied.

Conclusions: Heterogeneity in CYP2D6 gene rearrangement exists, but the allele frequency indicates that the risk for an erroneous phenotype prediction related to such variability is extremely low and that this risk can be neglected in routine analyses.
The Use of Wavelength‐selective Plastic Cladding Materials in Horticulture: Understanding of Crop and Fungal Responses Through the Assessment of Biological Spectral Weighting Functions
Authors: Paul Nigel D. , Jacobson Rob J. , Taylor Anna , Wargent Jason J. , Moore Jason P.
Published: April 2005

Plant responses to light spectral quality can be exploited to deliver a range of agronomically desirable end points in protected crops. This can be achieved using plastics with specific spectral properties as crop covers. We have studied the responses of a range of crops to plastics that have either (a) increased transmission of UV compared with standard horticultural covers, (b) decreased transmission of UV or (c) increased the ratio of red (R) : far‐red (FR) radiation. Both the UV‐transparent and R : FR increasing films reduced leaf area and biomass, offering potential alternatives to chemical growth regulators. The UV‐opaque film increased growth, but while this may be useful in some crops, there were tradeoffs with elements of quality, such as pigmentation and taste. UV manipulation may also influence disease control. Increasing UV inhibited not only the pathogenic fungus Botrytis cinerea but also the disease biocontrol agent Trichoderma harzianum. Unlike B. cinerea, T. harzianum was highly sensitive to UV‐A radiation. These fungal responses and those for plant growth in the growth room and the field under different plastics are analyzed in terms of alternative biological spectral weighting functions (BSWF). The role of BSWF in assessing general patterns of response to UV modification in horticulture is also discussed.
Effects of Size Structure on Trophic Interactions between Age‐0 Smallmouth Bass and Juvenile Anadromous Alewives
Authors: Hanson S. Dale , Curry R. Allen
Published: April 2005

Age‐0 alewivesAlosa pseudoharengusmay compete for food with age‐0 smallmouth bassMicropterus dolomieu, possibly resulting in declines to some smallmouth bass fisheries in the northeastern United States and Atlantic Canada. Such competition could dramatically influence the recruitment of smallmouth bass in this region because they are at their northern limit of distribution and age‐0 smallmouth bass must accumulate sufficient lipid reserves to survive the overwinter period. We examined the temporal patterns of diet and isotopes of carbon and nitrogen for age‐0 alewives and smallmouth bass in a natural lake and a reservoir where alewife escapement is managed. Multiple alewife spawnings occurred in the lake, and small‐bodied alewives were abundant throughout the summer. There was minimal diet overlap, age‐0 alewives became a significant component of the age‐0 smallmouth bass diet, and age‐0 bass grew larger in this system. In the reservoir, escapement of alewife spawners was truncated, and a single‐size age‐0 cohort was observed. Alewives were similar in size to age‐0 smallmouth bass, and by late summer their diets showed some overlap and δ13C and δ15N levels were similar. Both predation and the potential for competition existed for these species, and their interactions depended on body size relationships. This is an important consideration in alewife management strategies where coexisting fisheries for smallmouth bass are desired.
Fine‐Scale Behavioral Responses of Pacific Salmonid Smolts as They Encounter Divergence and Acceleration of Flow
Authors: Kemp Paul S. , Gessel Mike H. , Williams John G.
Published: April 2005

We assessed fine‐scale behavioral responses of the smolts of four Pacific salmonid species to open and constricted channels in a flume. Natural migrants encountered two geometrically similar parallel channels with different hydraulic conditions representing constricted and open treatments. Observation of route selection under alternate discharge scenarios provided evidence of behavioral choice by smolts. As expected, the majority of smolts passed through the open channel in a ratio consistent with flow. After controlling for the influence of flow, both initial channel selection and subsequent channel rejection was higher for the constricted channel; rejection was probably due to fish detecting an area of rapidly accelerating flow. The majority of smolts traveled downstream headfirst and faster than the mean midcolumn water velocity. Those that faced the flow passed at a slower rate and tended to select the open treatment. The few yearling Chinook salmon Oncorhynchus tshawytscha and coho salmon O. kisutch smolts that did not pass through the treatment channels, but held position within the flume, were larger than their conspecifics that passed downstream. Large steelhead (anadromous rainbow trout O. mykiss) smolts and subyearling Chinook salmon were more likely to pass the constricted channel than smaller fish. These results suggest that efforts to effectively guide fish with diversion structures will require understanding how the structures alter the local hydraulic environment and, thus, influence fish behavior.
A Target Strength and Length Relationship for Striped Bass and White Perch
Authors: Hartman K. J. , Nagy B. W.
Published: April 2005

For hydroacoustic studies in which estimation of fish size and absolute abundance is desirable, researchers must either develop a relationship between target strength and fish length or borrow one from the literature. The purpose of this study was to provide a relationship between target strength and total length for two economically and ecologically important sympatric species: striped bass Morone saxatilis and white perch M. americana. We measured the dorsal aspect target strength of 12 white perch and 22 striped bass with a 120‐kHz split‐beam hydroacoustic system. The calculated target strength (TS (decibels)) to length (Lcm (cm)) relationships were as follows: white perch TS = 26.48 log10Lcm − 69.45 (r2 = 0.81, P < 0.001); striped bass TS = 15.37 log10Lcm − 56.26 (r2 = 0.80, P < 0.001); and a combined model TS = 15.613 log10Lcm − 56.47 (r2 = 0.82, P < 0.001). Each of these models differs substantially from the equations of Love (1971) and Foote (1987), which are commonly used in acoustic studies when the actualrelationships are lacking.
Competitive Ability and Mortality of Growth‐Enhanced Transgenic Coho Salmon Fry and Parr When Foraging for Food
Authors: Vandersteen Tymchuk W. E. , Abrahams M. V. , Devlin R. H.
Published: April 2005

Coho salmon Oncorhynchus kisutch were genetically altered to produce growth hormone without regulation, causing them grow 11 times larger (on average) than control fish after 14 months. This technology has potential benefits for the aquaculture industry, but the environmental risk associated with the escape of transgenic fish into the wild is not known. To partially address this issue, we experimentally investigated how well transgenic salmon survive when given a choice to consume food in a predator's presence. If transgenic salmon are to retain their growth advantage, we predict that they must also be more effective at competing for food than wild salmon and be willing to suffer higher mortality rates when foraging. The relative competitive abilities of transgenic and control salmon at two different ages were tested with an unequal competitors ideal free distribution. A larger proportion of transgenic salmon fed within the system, although they were not overrepresented at a higher‐quantity food source. When feeding in the presence of a predator, there was no measurable difference in mortality rates between transgenic and control salmon at both the fry and parr stages. These data indicate that, under the limited environmental conditions we tested transgenic coho salmon are at least competitively equal to control fish and do not suffer higher rates of mortality to acquire food resources and maintain their enhanced growth.
Pathological and Clinical Response of a Primary Chemotherapy Regimen Combining Vinorelbine, Epirubicin, and Paclitaxel as Neoadjuvant Treatment in Patients with Operable Breast Cancer
Authors: Abrial Catherine , Van Praagh Isabelle , Delva Rémi , Leduc Bernard , Fleury Joël , Gamelin Erick , Sillet-Bach Isabelle , Penault-Llorca Frédérique , Amat Sophie , Chollet Philippe
Published: April 2005

This phase II study investigated the efficacy and tolerability of a primary chemotherapy regimen combining vinorelbine, epirubicin, and paclitaxel (VEP protocol) in women with stage II/III operable breast cancer. Patients (n = 50) were treated with six cycles of VEP according to the following schedule: vinorelbine (Navelbine®; Pierre Fabre, Boulogne, France; http://www.pierre-fabre.com) 20 mg/m2, epirubicin (Farmorubicin®; Pharmacia, New York, NY; http://www.pnu.com) 35 mg/m2 given on days 1 and 8, paclitaxel (Taxol®; Bristol-Myers Squibb, New York, NY; http://www.bmsoncology.com) 175 mg/m2 given on day 9, and G-CSF 5 mg/kg/day given on days 10–20 of a 21-day cycle, followed by surgery and radiotherapy. After six cycles of VEP, the pathological response rate (pCR) in breast was confirmed in six patients (12%; 95% confidence interval [CI]: 3–21)) using Chevallier's classification and in nine patients (18%; 95% CI: 7.4–28.6) using Sataloff's classification. The clinical response rate was 42% (95% CI: 28.3–55.7), including 26% complete responses. Breast conservation was achieved in 68% of patients. After a median follow-up of 48 months (range, 34–62 months), 16 relapses were observed. The overall and disease-free survivals at 5 years were 54.1% (95% CI: 40.3–67.9) and 38% (95% CI: 24.1–51.9), respectively. The principal toxicities of VEP were grade 3/4 neutropenia observed in 30% of patients and grade 3 anemia observed in 12% of patients. There was no case of severe cardiac toxicity, thrombocytopenia, or any other serious adverse events. In conclusion, whereas this regimen was relatively well tolerated, it appears inferior to other regimens and its use is not recommended.
Critical Evaluation of Current Treatments in Metastatic Colorectal Cancer
Authors: Venook Alan
Published: April 2005

Learning Objectives

After completing this course, the reader will be able to: Describe the evolution of cancer chemotherapy for patients with colorectal cancer.Discuss the current relevance of VEGF as a therapeutic target in colorectal cancer.Discuss the current relevance of EGFR as a therapeutic target in colorectal cancer.

Access and take the CME test online and receive 1 hour of AMA PRA category 1 credit at CME.TheOncologist.com

Fluorouracil (FU) has been the mainstay of treatment for metastatic colorectal cancer (mCRC) for many years. However, in recent years, newer chemotherapeutic agents, particularly irinotecan (Campostar®; Pfizer Pharmaceuticals, New York, NY, http://www.pfizer.com) and more recently oxaliplatin (Eloxatin®; Sanofi-Aventis Inc., New York, NY, http://www.sanofi-aventis.com), have been shown to improve survival in combination with FU-based therapies. These agents were therefore incorporated into first- and second-line treatment strategies. The development of targeted agents that are tumor specific with better toxicity profiles than chemotherapeutic agents has widened the spectrum of therapies for this disease. The U.S. Food and Drug Administration (FDA) recently approved two targeted agents for treating mCRC: an antivascular endothelial growth factor monoclonal antibody (mAb), bevacizumab (Avastin®; Genentech, Inc., South San Francisco, CA, http://www.gene.com), in combination with first-line 5-FU-based chemotherapy regimens and the human epidermal growth factor receptor (HER-1/EGFR)-targeted mAb cetuximab (Erbitux®; ImClone Systems, Inc., New York, NY, http://www.imclone.com) as monotherapy or in combination with irinotecan as second-line therapy in refractory cancer. These newer, more effective agents are improving clinical outcome for patients with mCRC. However, as the number of agents has increased, choosing the most effective treatment strategy has become increasingly complex. This review discusses the role of the individual agents in the treatment of mCRC and identifies the most effective regimens.
Liver Transplantation for Malignancy
Authors: Hertl Martin , Cosimi A. Benedict
Published: April 2005

Learning Objectives

After completing this course, the reader will be able to: List the indications for liver transplantation in patients with hepatobiliary malignancies.Describe the MELD and PELD system.Discuss the management and treatment algorithm of a patient with hepatobiliary malignancy.

Access and take the CME test online and receive 1 hour of AMA PRA category 1 credit at CME.TheOncologist.com

Liver transplantation for hepatic malignancies has emerged from an exotic and desperate approach to a well-documented and proven treatment modality for these unfortunate patients. However, early unsatisfactory results emphasized that only a highly selected patient population would benefit from transplantation. Currently, <10% of all liver transplants performed are for hepatocellular cancer (HCC). There is no controversy that hepatoblastoma is an excellent indication in pediatric patients with unresectable tumors. Similarly, liver transplantation for HCC in the adult population yields good results for patients whose tumor masses do not exceed the Milan criteria. It remains to be determined whether patients with more extensive tumors can be reliably selected to benefit from the procedure. Adjunctive procedures like radiofrequency ablation, chemoembolization, or cryotherapy might be indicated to limit tumor progression for patients on waiting lists. Epitheloid hemangioendothelioma is also an appropriate indication for liver transplantation, unlike angiosarcoma. Metastatic liver disease is not an indication for liver transplantation, with the exception of cases in which the primary is a neuroendocrine tumor, for which liver transplantation can result in long-term survival and even cure in a number of patients. And finally, while gallbladder cancers are never an indication for liver transplantation, rare cases of cholangiocellular cancer might qualify if aggressive combination therapies, including chemotherapy and radiotherapy followed by OLT, are carried through. Survival in these selected patients can approach that for patients with cholestatic liver disease.
Three Emerging New Drugs for NSCLC: Pemetrexed, Bortezomib, and Cetuximab
Authors: Dubey Sarita , Schiller Joan H.
Published: April 2005

Learning Objectives

After completing this course, the reader will be able to: Describe the targets of new biologic anticancer drugs.Interpret pharmacodynamic characteristics of drugs and apply this information in clinical use.Identify approved indications for the use of these new drugs.

Access and take the CME test online and receive 1 hour of AMA PRA category 1 credit at CME.TheOncologist.com

Despite advances made in cytotoxic chemotherapy, the prognosis for patients with non-small cell lung cancer (NSCLC) continues to be poor. New, more effective drugs must be identified and developed to improve the outcome of these patients. Three drugs with promising activity in NSCLC are pemetrexed (Alimta®; Eli Lilly and Company, Indianapolis, IN, http://www.lilly.com), bortezomib (Velcade®; Millennium Pharmaceuticals, Inc., Cambridge, MA, http://www.mlnm.com), and cetuximab (Erbitux®; ImClone Systems, Inc., New York, NY, http://www.imclone.com). Pemetrexed inhibits thymidylate synthase, dihydrofolate reductase, and glycinamide ribonucleotide formyl transferase, enzymes necessary for purine and pyrimidine synthesis, thus causing cell-cycle arrest in the S phase. Bortezomib, a proteasome inhibitor, interferes with the cytosolic protein degradation machinery, namely the ubiquitin-proteasome complex, causing breakdown of cell-cycle regulators and cell-cycle arrest. Cetuximab is a chimeric mouse-human antibody that inhibits ligand-dependent activation of the epidermal growth factor receptor, resulting in receptor internalization and inhibition of downstream pathways that, in turn, causes cell growth and progression. All three drugs are approved for different tumor types, and studies defining their role in NSCLC are under way.
Estrogen-related receptor-γ and peroxisome proliferator-activated receptor-γ coactivator-1α regulate estrogen-related receptor-α gene expression via a conserved multi-hormone response element
Authors: Liu D , Zhang Z , Teng C T
Published: April 2005

The expression of estrogen-related receptor-α (ERRα) is stimulated by estrogen in selective tissues. Recently, a correlation between ERRα expression and the induction of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) in the liver of fasting animals and in cold-stressed brown-fat tissues and skeletal muscle was shown. To explore the molecular mechanisms of ERRα regulation by diverse signals, the promoter of the human ERRα gene was cloned and characterized. Mutation and deletion analyses revealed that a 53 bp region containing repeated core element AGGTCA motifs of the ERRα gene serves as a multi-hormone response element (MHRE) for several nuclear receptors in transient co-transfection studies of human endometrial carcinoma (HEC-1B) cells. Among the nuclear receptors tested, ERRγ bound to and robustly stimulated the transcription of reporters containing at least two AGGTCA motifs. Ectopic expression of PGC-1α in HEC-1B cells strongly activated the reporter containing the MHRE, presumably via the endogenous nuclear receptor binding to the element. Reducing the endogenous level of ERRγ by small interfering RNA, and increasing the ERRγ level by ectopic expression, substantially decreased and increased respectively the transactivation capability of PGC-1α. The activation function 2 domain of the ERRγ and the L2 and L3 motifs of PGC-1α were essential to transactivate the MHRE. Additionally, PGC-1α increases the amount of endogenous ERRγ bound to the MHRE region as determined by a chromatin immunoprecipitation assay. The present study demonstrates that the MHRE of the ERRα gene is a target for ERRγ transactivation, which is enhanced by PGC-1α.
The acquisition of an insulin-secreting phenotype by HGF-treated rat pancreatic ductal cells (ARIP) is associated with the development of susceptibility to cytokine-induced apoptosis
Authors: Anastasi E , Santangelo C , Bulotta A , Dotta F , Argenti B , Mincione C , Gulino A , Maroder M , Perfetti R , Di Mario U
Published: April 2005

The elucidation of mechanisms regulating the regeneration and survival of pancreatic beta cells has fundamental implications in the cell therapy of type 1 diabetes. The present study had the following three aims: 1. to investigate whether pancreatic ductal epithelial cells can be induced to differentiate into insulin-producing cells by exposing them to hepatocyte growth factor (HGF); 2. to characterize some of the molecular events leading to their differentiation toward a beta-cell-like phenotype; 3. to evaluate the susceptibility of newly differentiated insulin-secreting cells to cytokine-induced apoptosis, a mechanism of beta-cell destruction occurring in type 1 diabetes. We demonstrated that HGF-treated rat pancreatic ductal cell line (ARIP) cells acquired the capability to transcribe the insulin gene and translate its counterpart protein. HGF-treated cells also exhibited a glucose-dependent capability to secrete insulin into the cultured medium. Expression analysis of some of the genes regulating pancreatic beta-cell differentiation revealed a time-dependent transcription of neurogenin-3 and Neuro-D in response to HGF. Finally, we determined the susceptibility to proinflammatory cytokine (PTh1)-induced apoptosis by incubating HGF-treated and untreated ARIP cells with a cocktail of interleukin-1 beta (IL-1β), tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ). Such treatment induced apoptotic death, as determined by the TUNEL technique, in about 40% of HGF-treated, insulin-secreting ARIP cells, while untreated ARIP cells were resistant to PTh1-induced apoptosis. In conclusion, we showed that HGF promotes the differentiation of ARIP cells into pancreatic beta-cell-like cells, and that the differentiation toward an insulin-secreting phenotype is associated with the appearance of susceptibility to cytokine-induced apoptosis.
Expression of the human melanocortin-4 receptor gene is controlled by several members of the Sp transcription factor family
Authors: Blondet A , Gout J , Durand P , Bégeot M , Naville D
Published: April 2005

The melanocortin-4 receptor (MC4-R) plays a key role in the hypothalamic control of food intake, lending importance to the understanding of the mechanisms that regulate its expression. To identify factors controlling the expression of the human (h) MC4-R gene, a fragment containing 1253 bp of the 5′-flanking region of the hMC4-R gene was isolated. A series of hMC4-R luciferase constructs were developed and used to transiently transfect HEK293 and GT1–7 cell lines, both expressing endogenous MC4-R mRNA. Deletion analysis of the 1253 bp fragment showed that the basal promoter activity is mainly restricted to the 179 bp upstream of the transcription start site in both cell types. Mutation of a putative Sp1-binding site located at position −76 bp resulted in a dramatic reduction of the luciferase activity in HEK293 and GT1–7 cells by 87 and 80% respectively. Both in vitro and in vivo studies (gel shift and chromatin immunoprecipitation analyses) revealed binding of both Sp1 and Sp3 to this site in HEK293 cells. Cotransfection with an Sp1 expression vector in Drosophila cells that do not express Sp1, in conjunction with treatment of HEK293 cells with mithramycin A, a specific inhibitor of Sp1, confirmed the role of Sp1. For the first time, we have demonstrated that the constitutive activity of the hMC4-R promoter is dependent upon Sp transcription factors.
Paracrine regulation of growth hormone gene expression by gonadotrophin release in grass carp pituitary cells: functional implications, molecular mechanisms and signal transduction
Authors: Zhou Hong , Jiang Yonghua , Ko Wendy K W , Li Wensheng , Wong Anderson O L
Published: April 2005

Growth hormone (GH) is known to stimulate luteinizing hormone (LH) release via paracrine interactions between somatotrophs and gonadotrophs. However, it is unclear if LH can exert a reciprocal effect to modulate somatotroph functions. Here we examined the paracrine effects of LH on GH gene expression using grass carp pituitary cells as a cell model. LH receptors were identified in grass carp somatotrophs and their activation by human chorionic gonadotropin (hCG) increased ‘steady-state’ GH mRNA levels. Removal of endogenous LH by immunoneutralization using LH antiserum inhibited GH release and GH mRNA expression. GH secretagogues, including gonadotrophin releasing hormone (GnRH), pituitary adenylate cyclase-activating polypeptide (PACAP) and apomorphine, were effective in elevating GH mRNA levels but these stimulatory actions were blocked by LH antiserum. In pituitary cells pretreated with actinomycin D, the half-life of GH mRNA was not affected by hCG but was enhanced by LH immunoneutralization. Treatment with LH antiserum also suppressed basal levels of mature GH mRNA and primary transcripts. hCG increased cAMP synthesis in carp pituitary cells and hCG-induced GH mRNA expression was mimicked by forskolin but suppressed by inhibiting adenylate cyclase and protein kinase A. Similarly, the stimulatory actions of hCG and forskolin on GH mRNA expression were blocked by inhibiting Janus kinase 2 (JAK2) and MAP kinase (MAPK), including P42/44MAPK and P38 MAPK. These results suggest that LH is essential for the maintenance of GH release, GH gene expression, and somatotroph responsiveness to GH-releasing factors. The paracrine actions of LH on GH mRNA expression are mediated by a concurrent increase in GH gene transcription and GH mRNA turnover, probably through JAK2/MAPK coupled to the cAMP-dependent pathway.
Tetracycline-regulated secretion of human (pro)insulin following plasmid-mediated transfection of human muscle
Authors: Wilson Magdalene O , Scougall Kathleen T , Ratanamart Jarupa , McIntyre Elizabeth A , Shaw James A M
Published: April 2005

Long-term secretion of insulin by host muscle following transduction with an insulin gene construct offers the potential of gene therapy for diabetes without immunosuppression. Clinical implementation will be dependent on proof of principle in human tissue and a system for safely regulating basal insulin levels. Liposomal co-transfection with a tetracycline-responsive wild type human preproinsulin (pTRE-hppI1) or mutant construct (pTRE-hppI4), in which PC2 and PC3 cleavage sites were altered to form tetrabasic consensus sites for furin, together with pTet-off (coding for a transactivating protein) was evaluated in the C2C12 mouse myoblast cell line and human myoblasts following establishment in primary culture. In the absence of tetracycline, (pro)insulin secretion in C2C12 and human myoblasts transfected with tetracycline-responsive hppI1 and hppI4 constructs was comparable to that following transfection with equivalent constructs under the control of a constitutively active cytomegaloviral promoter. Percentage processing to mature insulin was <5% in C2C12 and human myoblasts transfected with pTet-off/pTRE-hppI1 but >90% in C2C12 cells and 45–60% in human myoblasts on transfection with pTet-off/pTRE-hppI4. Incremental dose-responsive suppression of proinsulin secretion was demonstrated in C2C12 and human myoblasts expressing pTet-off/pTRE-hppI1 following incubation with tetracycline (0–100 μg/ml) for up to 72 h. Reversibility was confirmed following tetracycline withdrawal. Dose-responsive tetracycline-inducible repression of mature insulin secretion was confirmed in C2C12 cells following transfection with pTet-off/pTRE-hppI4. Regulation of human proinsulin biosynthesis and secretion has been attained in vivo following plasmid-mediated gene transfer to rat skeletal muscle and oral tetracycline administration. In conclusion, processing to mature insulin has been confirmed following plasmid-mediated gene transfer to human muscle in addition to in vitro- and in vivo-regulated human proinsulin secretion employing the safe and well-tolerated antibiotic, tetracycline.
Glucocorticoids suppress macrophage migration inhibitory factor (MIF) expression in a cell-type-specific manner
Authors: Alourfi Z , Donn R P , Stevens A , Berry A , McMaster A , Ray D W
Published: April 2005

MIF is a potent proinflammatory cytokine involved in inflammatory arthritis. Glucocorticoids (GC) have been reported to induce secretion of MIF in rodent cells, and as MIF counteracts the anti-inflammatory effects of GC, this has implications for human inflammatory disease. Transient transfection studies showed that the MIF promoter was repressed by dexamethasone (Dex) (10 nM) in CEM C7A cells, with up to 50% suppression by 100 nM. However, there was no regulation of the promoter by GC in A549 cells. We also found that subnanomolar concentrations of Dex suppressed MIF secretion, measured by ELISA, by 80% in both human T lymphoblasts (CEM C7A) and human lung epithelial cells (A549). Endogenous MIF mRNA was also repressed by GC in CEM C7A cells, measured both by Northern blot and quantitative RT-PCR assays, but there was no such regulation in A549 cells. This suggests that GC affects translation rather than transcription of MIF in A549 cells. These results contradict earlier results with the rat cell line RAW 264.7. Therefore, we analysed MIF secretion from RAW 264.7 cells but found no GC effect on secretion. Understanding how GC regulates MIF in a cell-type-dependent manner may give insights into GC-refractory human inflammatory diseases.
Pancreatic β-cells expressing GLP-1 are resistant to the toxic effects of immunosuppressive drugs
Authors: D’Amico Eugenio , Hui Hongxiang , Khoury Nasif , Di Mario Umberto , Perfetti Riccardo
Published: April 2005

Glucose intolerance is often observed after pancreatic islet cell transplantation. The administration of immunosuppressive agents (ISD), necessary to avoid tissue rejection, is in part responsible for hyperglycemia. To investigate whether mouse insulinoma (MIN6) cells transfected with the glucagon like peptide-1 (GLP-1) fragment of the proglucagon gene (RIP/GLP-1 MIN6 cells) are resistant to the toxicity derived from the administration of ISD. RIP/GLP-1 MIN6 cells, as well as parental MIN6 cells, were exposed to a cocktail of ISD. The secretion of insulin and the expression of apoptosis-related proteins were investigated by RIA and western blot analysis. Cell apoptosis was quantified by FACS analysis. Finally, to study whether the antiapoptotic action of GLP-1 was a function of its effect on insulin secretion, or rather it was a direct effect of GLP-1, cells were cultured with or without diazoxide or exendin-9. GLP-1 improved the functional activity and the viability of cells exposed to ISD. The insulin secretion of RIP/GLP-1 MIN6 cells after exposure to ISD was preserved. The expression of GLP-1 by β-cells reduced the number of apoptotic cells and increased the expression of the antiapoptotic protein Bcl-2. GLP-1 also decreased the abundance of the proapoptotic markers PARP-p85 and Smac/Diablo. Treatment of cells with the diazoxide did not abolish the protective advantage that cells transfected with GLP-1 had; conversely the exposure of cells to exendin-9 was associated with a restored susceptibility to apoptosis. This report demonstrates that GLP-1 is capable of preserving β-cell function and protecting cells from apoptotic cell death.
βA- and βC-activin, follistatin, activin receptor mRNA and βC-activin peptide expression during rat liver regeneration
Authors: Gold E J , Zhang X , Wheatley A M , Mellor S L , Cranfield M , Risbridger G P , Groome N P , Fleming J S
Published: April 2005

The mRNA expression of two activin growth factor subunits (βA- and βC-activin), activin receptor subunits (ActRIIA, ActRIIB) and the activin-binding protein follistatin, and peptide expression of βA-activin and βC-activin subunits, were examined in regenerating rat liver after partial hepatectomy (PHx). Liver samples were collected from adult, male Sprague–Dawley rats, 12–240 h (n=3–5 rats per time point) after PHx or from sham-operated controls at the same time points. Hepatocyte mitosis and apoptosis were assessed histologically and by in situ cell death detection. RT and PCR were used to assess relative gene expression. βA- and βC-activin peptide immunoreactivity was assessed in liver and serum samples by western blotting, whereas cellular expression was investigated by immunohistochemistry, using specific monoclonal antibodies. βA- and βC-activin mRNA dropped to < 50% of sham control values 12 h after PHx and remained at this level until 168 h post-PHx, when βA-activin expression increased to three times sham control values and βC-activin mRNA returned to pre-PHx levels. A peak in follistatin expression was observed 24–48 h post-PHx, coincident with an increase in hepatocyte mitosis. No changes were observed in ActRIIA mRNA, whereas ActRIIB expression paralleled that of βA-activin mRNA. βC-activin immunoreactive homo- and heterodimers were observed in regenerating liver and serum. Mitotic hepatocytes frequently contained βC-activin immunoreactivity, whereas apoptotic hepatocytes were often immunoreactive for βA-activin. We conclude that βA- and βC-activin subunit proteins are autocrine growth regulators in regenerating liver and when expressed independently lead to hepatocyte apoptosis or mitosis in a subset of hepatocytes.
Salivary leptin induces increased expression of growth factors in oral keratinocytes
Authors: Gröschl M , Topf H-G , Kratzsch J , Dötsch J , Rascher W , Rauh M
Published: April 2005

We investigated the influence of salivary leptin, purified by affinity chromatography, on the proliferation of human oral keratinocytes. Furthermore we determined the time- and dose-dependency of the incubation with salivary leptin on the production of epidermal growth factor (EGF) and keratinocyte growth factor (KGF), which are growth factors relevant to keratinocyte proliferation. The analysis was performed both intra- and extracellularly. The relationship between the three cytokines in cell proliferation was studied by successive blocking with specific antibodies. The incubation of oral keratinocytes with recombinant and native leptin led to a significantly increased, dose-dependent cell proliferation (P<0.001). A further significant increase in proliferation was observed after incubating the cells with sterile filtered saliva (P<0.001). The increase in proliferation could not be observed by simultaneous incubation with salivary leptin and specific antibodies against either leptin or KGF (P<0.001). We found a significant dose-dependency between leptin incubation and production of KGF and EGF at the RNA and protein level. Both cytokines were expressed intracellularly and released into the culture medium, where they could be quantified by ELISA. Furthermore, there was a dose- and time-dependent increase in the phosphorylation of STAT-1 and STAT-3, indicating that Ob-Rb (the long form of the leptin receptor) expressed by the keratinocytes is functional. It is conceivable that the leptin-induced proliferation in keratinocytes is mediated by this signalling pathway. This is the first study to show a physiological role of salivary leptin as a growth factor for keratinocyte proliferation in the oral cavity. We could demonstrate its influence on the production of other growth factors essential for this proliferative effect. Based on the findings of our study we assume an important role for salivary leptin in wound healing within the vulnerable oral cavity.
Gonadal defects and hormonal alterations in transgenic mice expressing a single chain human chorionic gonadotropin–lutropin receptor complex
Authors: Meehan Thomas P , Harmon Barry G , Overcast Megan E , Yu Kristine K , Camper Sally A , Puett David , Narayan Prema
Published: April 2005

To study the effects of premature and chronic ligand-mediated luteinizing hormone receptor (LHR) activation on reproductive development, we have generated transgenic mice expressing a genetically engineered, constitutively active yoked hormone–receptor complex (YHR), in which a fusion protein of human chorionic gonadotropin (hCG) is covalently linked to the N-terminus of rat LHR. YHR-expressing mice (YHR+) were analyzed at pre- and post-pubertal ages. Relative to wild type (WT) controls, male mice exhibited prepubertal increases in testosterone levels and seminal vesicle weights, and decreases in serum FSH, serum LH, testes weight, and the size of the seminiferous tubules. In adult male YHR+ mice, testosterone and LH levels are not significantly different from WT controls. However, FSH levels and testes weights remain decreased. Female YHR+ mice undergo precocious puberty with early vaginal opening, accelerated uterine development, enhanced follicular development, including the presence of corpora lutea, and an increase in serum progesterone. At 12 weeks of age, the ovary exhibits a relative increase in the amount of interstitial tissue, comprised of cells that are hypertrophic and luteinized, as well as follicles that are degenerating. Additionally, hemorrhagic cysts develop in approximately 25% of the transgenic mice. These degenerative changes are consistent with an aging ovary suggesting that CG-induced LHR activation in female mice leads to precocious sexual development and ovarian lesions. Taken together, these data indicate that the single chain YHR is functional in vivo and demonstrate that YHR+ mice provide a novel system to further understand the reproductive consequences of aberrant LHR activation.
Analysis of gene expression profiles in insulin-sensitive tissues from pre-diabetic and diabetic Zucker diabetic fatty rats
Authors: Suh Young Ho , Kim Younyoung , Bang Jeong Hyun , Choi Kyoung Suk , Lee June Woo , Kim Won-Ho , Oh Tae Jeong , An Sungwhan , Jung Myeong Ho
Published: April 2005

Insulin resistance occurs early in the disease process, preceding the development of type 2 diabetes. Therefore, the identification of molecules that contribute to insulin resistance and leading up to type 2 diabetes is important to elucidate the molecular pathogenesis of the disease. To this end, we characterized gene expression profiles from insulin-sensitive tissues, including adipose tissue, skeletal muscle, and liver tissue of Zucker diabetic fatty (ZDF) rats, a well characterized type 2 diabetes animal model. Gene expression profiles from ZDF rats at 6 weeks (pre-diabetes), 12 weeks (diabetes), and 20 weeks (late-stage diabetes) were compared with age- and sex-matched Zucker lean control (ZLC) rats using 5000 cDNA chips. Differentially regulated genes demonstrating > 1.3-fold change at age were identified and categorized through hierarchical clustering analysis. Our results showed that while expression of lipolytic genes was elevated in adipose tissue of diabetic ZDF rats at 12 weeks of age, expression of lipogenic genes was decreased in liver but increased in skeletal muscle of 12 week old diabetic ZDF rats.

These results suggest that impairment of hepatic lipogenesis accompanied with the reduced lipogenesis of adipose tissue may contribute to development of diabetes in ZDF rats by increasing lipogenesis in skeletal muscle. Moreover, expression of antioxidant defense genes was decreased in the liver of 12-week old diabetic ZDF rats as well as in the adipose tissue of ZDF rats both at 6 and 12 weeks of age. Cytochrome P450 (CYP) genes were also significantly reduced in 12 week old diabetic liver of ZDF rats. Genes involved in glucose utilization were downregulated in skeletal muscle of diabetic ZDF rats, and the hepatic gluconeogenic gene was upregulated in diabetic ZDF rats. Genes commonly expressed in all three tissue types were also observed. These profilings might provide better fundamental understanding of insulin resistance and development of type 2 diabetes.
Mechanism of protein tyrosine phosphatase 1B-mediated inhibition of leptin signalling
Authors: Lund I K , Hansen J A , Andersen H S , Møller N P H , Billestrup N
Published: April 2005

Upon leptin binding, the leptin receptor is activated, leading to stimulation of the JAK/STAT signal transduction cascade. The transient character of the tyrosine phosphorylation of JAK2 and STAT3 suggests the involvement of protein tyrosine phosphatases (PTPs) as negative regulators of this signalling pathway. Specifically, recent evidence has suggested that PTP1B might be a key regulator of leptin signalling, based on the resistance to diet-induced obesity and increased leptin signalling observed in PTP1B-deficient mice. The present study was undertaken to investigate the mechanism by which PTP1B mediates the cessation of the leptin signal transduction. Leptin-induced activation of a STAT3 responsive reporter was dose-dependently inhibited by co-transfection with PTP1B. No inhibition was observed when a catalytically inactive mutant of PTP1B was used or when other PTPs were co-transfected. PTP1B was able to dephosphorylate activated JAK2 and STAT3 in vitro, whereas either no or a minimal effect was observed with cluster of differentiation 45 (CD45), PTPα and leukocyte antigen-related (LAR). By utilisation of a selective PTP1B inhibitor, the leptin-induced STAT3 activation was enhanced in cells. In conclusion, these results suggested that the negative regulatory role of PTP1B on leptin signalling is mediated through a direct and selective dephosphorylation of the two signalling molecules, JAK2 and STAT3.
Effect of inactivating mutations on phosphorylation and internalization of the human VPAC2 receptor
Authors: Langer Ingrid , Langlet Christelle , Robberecht Patrick
Published: April 2005

The VPAC2 receptor, as all members of the G-protein-coupled receptor (GPCR)-B family, has two highly conserved motifs in the third intracellular (IC3) loop: a lysine and a leucine located at the amino-terminus and two basic residues separated by a leucine and an alanine at the carboxyl-terminus. This study evaluates the involvement of those conserved amino acid sequences in VPAC2 signal transduction and regulation. The residues were mutated into alanine and mutants were expressed in Chinese hamster ovary (CHO) cells stably transfected with Gα16 and aequorin. Mutation of L310 reduced efficacy of vasoactive intestinal polypeptide (VIP) to stimulate adenylate cyclase activity through Gαs coupling by 75%, without affecting VIP capability to stimulate an increase in [Ca2+]i through Gα16 coupling. Mutation of R325 and, to a lesser extend, K328 reduced VIP efficacy to stimulate [Ca2+]i increase and VIP potency to stimulate adenylate cyclase. The combination of mutations of both amino- and carboxyl-terminus located conserved motifs of the IC3 loop generates an inactive receptor with respect to [Ca2+]i increase and adenylate cyclase activation, but also with respect to receptor phosphorylation and internalization that were indeed directly correlated with the potency of inactivation of the receptors.

The amino-terminus of the VPAC2 receptor IC3 loop is thus involved in adenylate cyclase activation and the carboxyl-terminus of the IC3 loop participates in both Gαs and Gα16 coupling. The mutations studied also reduced both receptor phosphorylation and internalization in a manner that appeared directly linked to the alteration of Gαs and Gα16 coupling.
The post-endocytotic fate of the gonadotropin receptors is an important determinant of the desensitization of gonadotropin responses
Authors: Bhaskaran R S , Ascoli M
Published: April 2005

Internalization of the ligand/receptor complexes is a consequence of the activation of the gonadotropin receptors. Since the recycling or degradation of the internalized receptors results in the maintenance or loss of cell surface receptors respectively and this contributes to the loss of responsiveness, we hypothesized that the fate of the internalized receptors could be an important component of desensitization. We examined this hypothesis using the wild-type and mutants of the human LH (hLHR) receptors and follitropin receptors expressed in MA-10 and KK-1 cells respectively. The receptor mutants were chosen because they are routed mostly to a lysosomal degradation pathway whereas the wild-type receptors are recycled back to the surface. We have shown that agonist stimulation of cells expressing the mutant receptors results in a more pronounced loss of cell surface receptors and agonist responses than stimulation of cells expressing the wild-type receptors. We concluded that receptor recycling promotes the maintenance of cell surface receptors and preserves hormonal responsiveness. This property of the hLHR is likely to be physiologically important because there at least two hLHR-expressing tissues in pregnant women, the maternal corpus luteum and the fetal Leydig cells, where a loss of hormonal responsiveness induced by the elevated levels of human chorionic gonadotropin that occur during pregnancy is not desirable.
How closely do acute lethal concentration estimates predict effects of toxicants on populations?
Authors: Stark John D.
Published: April 2005

Acute lethal dose/concentration estimates are the most widely used measure of toxicity and these data often are used in ecological risk assessment. However, the value of the lethal concentration (LC50) as a toxicological endpoint for use in ecological risk assessment recently has been criticized. A question that has been asked frequently is how accurate is the LC50 for prediction of longer-term effects of toxicants on populations of organisms? To answer this question, Daphnia pulex populations were exposed to nominal concentrations equal to the 48-h acute LC50 of 6 insecticides, Actara, Aphistar diazinon, pymetrozine, Neemix, and Spinosad; and 8 agricultural adjuvants, Bond, Kinetic, Plyac, R-11, Silwet, Sylgard 309, Water Maxx, and X-77; for 10 d. None of the D. pulex populations exposed to the acute LC50 of these insecticides were 50% lower than the control populations at the end of the study; exposure to diazinon resulted in populations that were higher than expected (91% of the control). Exposure to Actara and Aphistar resulted in populations that were <1 and 29% of the control, respectively. Exposure to Fulfill, Neemix, and Spinosad resulted in extinction. Extinction occurred after exposure to all of the adjuvants, except Silwet L-77 where the population was 31% of the control. These results corroborate other studies that indicate that the LC50 is not a good predictor of effects on population growth. Although lethal concentration estimates have their place in toxicology, namely to compare intrinsic toxicity of chemicals among species or susceptibility of a species to different chemicals over short time periods, population growth and growth-rate studies are necessary to predict toxicant effects on populations.