bluuebunny/crossref_metadata_2025 huggingface dataset:
Peptide-Lipid Huge Toroidal Pore, a New Antimicrobial Mechanism Mediated by a Lactococcal Bacteriocin, Lacticin Q
Authors: Yoneyama Fuminori , Imura Yuichi , Ohno Kanako , Zendo Takeshi , Nakayama Jiro , Matsuzaki Katsumi , Sonomoto Kenji
Published: May 2009

Lacticin Q is a pore-forming bacteriocin produced by
Lactococcus lactis
QU 5, and its antimicrobial activity is in the nanomolar range. Lacticin Q induced calcein leakage from negatively charged liposomes. However, no morphological changes in the liposomes were observed by light scattering. Concomitantly with the calcein leakage, lacticin Q was found to translocate from the outer to the inner leaflet of the liposomes, after it initially bound to the membrane within 2 s. Lacticin Q also induced lipid flip-flop. These results reveal that the antimicrobial mechanism of lacticin Q can be described by the toroidal pore model. This is the first report of a bacteriocin of gram-positive bacteria that forms a toroidal pore. From liposomes, lacticin Q leaked fluorescence-labeled dextran with a diameter of 4.6 nm. In addition, lacticin Q caused the leakage of small proteins, such as the green fluorescent protein, from live bacterial cells. There are no other reports of antimicrobial peptides that exhibit protein leakage properties. The proposed pore formation model of lacticin Q is as follows: (i) quick binding to outer membrane leaflets; (ii) the formation of at least 4.6-nm pores, causing protein leakage with lipid flip-flop; and (iii) the migration of lacticin Q molecules from the outer to the inner membrane leaflets. Consequently, we termed the novel pore model in the antimicrobial mechanism of lacticin Q a “huge toroidal pore.”
Telavancin Disrupts the Functional Integrity of the Bacterial Membrane through Targeted Interaction with the Cell Wall Precursor Lipid II
Authors: Lunde Christopher S. , Hartouni Stephanie R. , Janc James W. , Mammen Mathai , Humphrey Patrick P. , Benton Bret M.
Published: May 2009

Telavancin is an investigational lipoglycopeptide antibiotic currently being developed for the treatment of serious infections caused by gram-positive bacteria. The bactericidal action of telavancin results from a mechanism that combines the inhibition of cell wall synthesis and the disruption of membrane barrier function. The purpose of the present study was to further elucidate the mechanism by which telavancin interacts with the bacterial membrane. A flow cytometry assay with the diethyloxacarbocyanine dye DiOC
2
(3) was used to probe the membrane potential of actively growing
Staphylococcus aureus
cultures. Telavancin caused pronounced membrane depolarization that was both time and concentration dependent. Membrane depolarization was demonstrated against a reference
S. aureus
strain as well as phenotypically diverse isolates expressing clinically important methicillin-resistant (MRSA), vancomycin-intermediate (VISA), and heterogeneous VISA (hVISA) phenotypes. The cell wall precursor lipid II was shown to play an essential role in telavancin-induced depolarization. This was demonstrated both in competition binding experiments with exogenous
d
-Ala-
d
-Ala-containing ligand and in experiments with cells expressing altered levels of lipid II. Finally, monitoring of the optical density of
S. aureus
cultures exposed to telavancin showed that cell lysis does not occur during the time course in which membrane depolarization and bactericidal activity are observed. Taken together, these data indicate that telavancin's membrane mechanism requires interaction with lipid II, a high-affinity target that mediates binding to the bacterial membrane. The targeted interaction with lipid II and the consequent disruption of both peptidoglycan synthesis and membrane barrier function provide a mechanistic basis for the improved antibacterial properties of telavancin relative to those of vancomycin.
The
Acinetobacter baumannii
19606 OmpA Protein Plays a Role in Biofilm Formation on Abiotic Surfaces and in the Interaction of This Pathogen with Eukaryotic Cells
Authors: Gaddy Jennifer A. , Tomaras Andrew P. , Actis Luis A.
Published: May 2009

The ability of
Acinetobacter baumannii
to adhere to and persist on surfaces as biofilms could be central to its pathogenicity. The production of pili and a biofilm-associated protein and the expression of antibiotic resistance are needed for robust biofilm formation on abiotic and biotic surfaces. This multistep process also depends on the expression of transcriptional regulatory functions, some of which could sense nutrients available to cells. This report extends previous observations by showing that although outer membrane protein A (OmpA) of
A. baumannii
19606 plays a partial role in the development of robust biofilms on plastic, it is essential for bacterial attachment to
Candida albicans
filaments and A549 human alveolar epithelial cells. In contrast to abiotic surfaces, the interaction with biotic surfaces is independent of the CsuA/BABCDE-mediated pili. The interaction of
A. baumannii
19606 with fungal and epithelial cells also results in their apoptotic death, a response that depends on the direct contact of bacteria with these two types of eukaryotic cells. Furthermore, the bacterial adhesion phenotype correlates with the ability of bacteria to invade A549 epithelial cells. Interestingly, the killing activity of cell-free culture supernatants proved to be protease and temperature sensitive, suggesting that its cytotoxic activity is due to secreted proteins, some of which are different from OmpA.
The Multiplicity of Infection of a Plant Virus Varies during Colonization of Its Eukaryotic Host
Authors: González-Jara Pablo , Fraile Aurora , Canto Tomás , García-Arenal Fernando
Published: May 2009

The multiplicity of infection (MOI), i.e., the number of virus genomes that infect a cell, is a key parameter in virus evolution, as it determines processes such as genetic exchange among genomes, selection intensity on viral genes, epistatic interactions, and the evolution of multipartite viruses. In fact, the MOI level is equivalent to the virus ploidy during genome expression. Nevertheless, there are few experimental estimates of MOI, particularly for viruses with eukaryotic hosts. Here we estimate the MOI of
Tobacco mosaic virus
(TMV) in its systemic host,
Nicotiana benthamiana
. The progress of infection of two TMV genotypes, differently tagged with the green or red fluorescent proteins GFP and RFP, was monitored by determining the number of leaf cell protoplasts that showed GFP, RFP, or GFP and RFP fluorescence at different times postinoculation. This approach allowed the quantitative analysis of the kinetics of infection and estimation of the generation time and the number of infection cycles required for leaf colonization. MOI levels were estimated from the frequency of cells infected by only TMV-GFP or TMV-RFP. The MOI was high, but it changed during the infection process, decreasing from an initial level of about 6 to a final one of 1 to 2, with most infection cycles occurring at the higher MOI levels. The decreasing MOI can be explained by mechanisms limiting superinfection and/or by genotype competition within double-infected cells, which was shown to occur in coinfected tobacco protoplasts. To our knowledge, this is the first estimate of MOI during virus colonization of a eukaryotic host.
Call for a European programme in external quality assurance for bone marrow immunohistochemistry; report of a European Bone Marrow Working Group pilot study
Authors: Torlakovic E E , Naresh K , Kremer M , van der Walt J , Hyjek E , Porwit A
Published: May 2009

Background and Aims:

In diagnostic immunohistochemistry (IHC), daily quality control/quality assurance measures (QC/QA) and participation in external quality assurance programmes (EQA) are important in ensuring good laboratory practice and patient care. Bone marrow trephine biopsies (BMTB) have been generally excluded from EQA programmes for diagnostic IHC due to a lack of standards for tissue processing. The European Bone Marrow Working Group (EBMWG) has set up an EBMWG IHC Committee with the task of exploring the plausibility of an EQA programme for BMTB IHC in Europe.

Methods:

28 laboratories participated in a web-based anonymous survey; 19 laboratories submitted a total of 109 slides stained for CD34, CD117, CD20, CD3, Ki-67 and a megakaryocyte marker of choice.

Results:

Eight different fixatives and nine different decalcification methods were used. While 93% of participants believed that they produced excellent results in BMTB IHC, only 4/19 (21%) laboratories did not have any poor results. CD117 and Ki-67, with 53% and 50% poor results, respectively, were the most problematic immunostains, while CD20 was the least problematic, with only 11% poor results.

Conclusions:

The EBMWG IHC Committee calls for a reduction in the tissue processing methods for BMTB and establishment of an EQA programme for BMTB IHC to help diagnostic IHC laboratories calibrate their tests according to expert recommendations. This is especially necessary in the light of recent introduction of predictive IHC tests in BMTB.
Spindle cell tumour with glandular elements: an unusual ileal neoplasm
Authors: Bateman A C , Holgate K , Green B , Thway K , Primrose J
Published: May 2009

This report describes a tumour in the ileum with clinical features initially suggestive of a gastrointestinal stromal tumour (GIST). Histopathological examination revealed a biphasic tumour in which the spindle cell component showed immunohistochemical evidence of smooth muscle differentiation but without the characteristic profile of a GIST. A well-differentiated epithelial component was also present, comprising glandular structures with immunohistochemical features suggestive of Mullerian differentiation. Similar glandular differentiation has been described in uterine leiomyomas but not, to our knowledge, in tumours associated with the small bowel. None of the characteristic mutations of GISTs were identified in this case. There were no overt features of malignancy but, because of the unusual nature of the case, we assessed the biological behaviour as uncertain.
Relative effects of estrogen, age, and visceral fat on pulsatile growth hormone secretion in healthy women
Authors: Veldhuis Johannes D. , Hudson Susan B. , Erickson Dana , Bailey Joy N. , Reynolds George Ann , Bowers Cyril Y.
Published: May 2009

Growth hormone (GH) secretion is subject to complex regulation. How pre- and postmenopausal age (PRE, POST), estradiol (E2) availability, and abdominal visceral fat (AVF) jointly affect peptidyl-secretagogue drive of GH secretion is not known. To this end, healthy PRE ( n = 20) and POST ( n = 22) women underwent a low- vs. high-E2clamp before receiving a continuous intravenous infusion of GH-releasing hormone (GHRH) or GH-releasing peptide (GHRP-2). According to analysis of covariance, PRE and POST women achieved age-independent hypo- and euestrogenemia under respective low- and high-E2clamps. All four of age ( P < 0.001), E2status ( P = 0.006), secretagogue type ( P < 0.001), and an age × peptide interaction ( P = 0.014) controlled pulsatile GH secretion. Independently of E2status, POST women had lower GH responses to both GHRH ( P = 0.028) and GHRP-2 ( P < 0.001) than PRE women. Independently of age, GHRP-2 was more stimulatory than GHRH during low E2( P = 0.011) and high E2( P < 0.001). Stepwise forward-selection multivariate analysis revealed that computerized tomographic estimates of AVF explained 22% of the variability in GHRH action ( P = 0.002), whereas age and E2together explained 60% of the variability in GHRP-2 drive ( P < 0.001). These data establish that age, estrogen status, and AVF are triple covariates of continuous peptide-secretagogue drive of pulsatile GH secretion in women. Each factor must be controlled for to allow valid comparisons of GH-axis activity.
Decreased Jun-D and myogenin expression in muscle wasting of human cachexia
Authors: Ramamoorthy Sonia , Donohue Michael , Buck Martina
Published: May 2009

Muscle wasting is a critical feature of patients afflicted by acquired immune deficiency syndrome (AIDS), cancer, or chronic inflammatory diseases. In a mouse model of muscle wasting, TNF-α induces oxidative stress and nitric oxide synthase-2 (NOS2) and decreases myogenin, Jun-D, and creatinine kinase muscle isoform (CKM) expression. Here, we studied 12 patients with muscle wasting due to cancer ( N = 10) or AIDS ( N = 2) and 4 control subjects. We show that in skeletal muscle of cachectic patients there is 1) increased expression and activity of the TNF-α signaling, including TNF-α mRNA, activation of TNFR1, and TNF-α-associated to TNFR1; 2) increased oxidative stress, as determined by the presence of malondialdehyde-lysine adducts; 3) increased NOS2 mRNA and protein; 4) decreased expression of Jun-D, myogenin, myosin, and CKM mRNA and protein; 5) impaired CKM-E box binding activities, associated with decreased Jun-D/myogenin activities; and 6) oxidative modification and ubiquitination of Jun-D. These studies show that these molecular pathways are modulated in association with muscle wasting in patients with cancer or AIDS, and whether or not they cause muscle wasting remains to be determined.
Overexpression of follistatin in trout stimulates increased muscling
Authors: Medeiros Erika F. , Phelps Michael P. , Fuentes Fernando D. , Bradley Terence M.
Published: May 2009

Deletion or inhibition of myostatin in mammals has been demonstrated to markedly increase muscle mass by hyperplasia, hypertrophy, or a combination of both. Despite a remarkably high degree of conservation with the mammalian protein, the function of myostatin remains unknown in fish, many species of which continue muscle growth throughout the lifecycle by hyperplasia. Transgenic rainbow trout ( Oncorhynchus mykiss) overexpressing follistatin, one of the more efficacious antagonists of myostatin, were produced to investigate the effect of this protein on muscle development and growth. P1 transgenics overexpressing follistatin in muscle tissue exhibited increased epaxial and hypaxial muscling similar to that observed in double-muscled cattle and myostatin null mice. The hypaxial muscling generated a phenotype reminiscent of well-developed rectus abdominus and intercostal muscles in humans and was dubbed “six pack.” Body conformation of the transgenic animals was markedly altered, as measured by condition factor, and total muscle surface area increased. The increased muscling was due almost exclusively to hyperplasia as evidenced by a higher number of fibers per unit area and increases in the percentage of smaller fibers and the number of total fibers. In several individuals, asymmetrical muscling was observed, but no changes in mobility or behavior of follistatin fish were observed. The findings indicate that overexpression of follistatin in trout, a species with indeterminate growth rate, enhances muscle growth. It remains to be determined whether the double muscling in trout is due to inhibition of myostatin, other growth factors, or both.
Coherence Between Motor Cortical Activity and Peripheral Discontinuities During Slow Finger Movements
Authors: Williams Elizabeth R. , Soteropoulos Demetris S. , Baker Stuart N.
Published: May 2009

Slow finger movements in man are not smooth, but are characterized by 8- to 12-Hz discontinuities in finger acceleration thought to have a central source. We trained two macaque monkeys to track a moving target by performing index finger flexion/extension movements and recorded local field potentials (LFPs) and spike activity from the primary motor cortex (M1); some cells were identified as pyramidal tract neurons by antidromic activation or as corticomotoneuronal cells by spike-triggered averaging. There was significant coherence between finger acceleration in the approximately 10-Hz range and both LFPs and spikes. LFP–acceleration coherence was similar for flexion and extension movements (0.094 at 9.8 Hz and 0.11 at 6.8 Hz, respectively), but substantially smaller during steady holding (0.0067 at 9.35 Hz). The coherence phase showed a significant linear relationship with frequency over the 6- to 13-Hz range, as expected for a constant conduction delay, but the slope indicated that LFP lagged acceleration by 18 ± 14 or 36 ± 8 ms for flexion and extension movements, respectively. Directed coherence analysis supported the conclusion that the dominant interaction was in the acceleration to LFP (i.e., sensory) direction. The phase relationships between finger acceleration and both LFPs and spikes shifted by about π radians in flexion compared with extension trials. However, for a given trial type the phase relationship with acceleration was similar for cells that increased their firing during flexion or during extension trials. We conclude that movement discontinuities during slow finger movements arise from a reciprocally coupled network, which includes M1 and the periphery.
Antibacterial Properties of Nanosilver PLLA Fibrous Membranes
Authors: Li Lin , Li Yi , Li Jiashen , Yao Lei , Mak Arthur F. T. , Ko Frank , Qin Ling
Published: May 2009

Nanosilver has been studied as a valuable material for it strong antibacterial effects. In this study, we investigated the antibacterial properties of nano silver Poly‐L‐Lactic acid (Ag/PLLA) composite fibrous membranes. Ag/PLLA fibrous membranes were prepared with silver nanoparticles having weight ratio of silver nanoparticles to PLLA at 5% (w/w). In vitro antibacterial tests were performed using Escherichia coli (E. coli) and
Staphylococcus aureus (Staph.) to determine the antibacterial capability of the Ag/PLLA fibrous membranes. As the results suggested, Ag/PLLA fibrous membranes showed strong antibacterial properties. Thus, Ag/PLLA fibrous membrane can be used as an antibacterial scaffold for tissue engineering.
Could Proteomic Research Deliver the Next Generation of Treatments for Pneumococcal Meningitis?
Authors: Goonetilleke U. R. , Ward S. A. , Gordon S. B.
Published: May 2009

Streptococcus pneumoniaeis the most common bacterial cause of community-acquired meningitis worldwide. Despite optimal antibiotic therapy and supportive care, the mortality of this condition remains very high at 20–30% in the developed world and over 60% in under-resourced hospitals. In developed countries, approximately half of the survivors suffer intellectual impairment, hearing loss, or other neurological damage. There is an urgent need for more information about the mechanisms of brain damage and death in pneumococcal meningitis so that new treatments can be designed. Using proteomic techniques and bioinformatics, the protein content of cerebrospinal fluid can be examined in great detail. Animal models have added greatly to our knowledge of possible mechanisms and shown that hippocampal apoptosis and cortical necrosis are distinct mechanisms of neuronal death. The contribution of these pathways to human disease is unknown. Using proteomic techniques, neuronal death pathways could be described in CSF samples. This information could lead to the design of novel therapies to minimize brain damage and lower mortality. This minireview will summarize the known pathogenesis of meningitis, and current gaps in knowledge, that could be filled by proteomic analysis.
Interleukin-23 and Th17 Cells in the Control of Gut Inflammation
Authors: Monteleone Ivan , Pallone Francesco , Monteleone Giovanni
Published: May 2009

Crohn's Disease and Ulcerative Colitis, the major forms of inflammatory bowel diseases (IBDs) in humans, have been traditionally associated with exaggerated and poorly controlled T helper (Th) type 1 or Th2 cell response, respectively. More recent studies have, however, shown that IBDs are also characterized by a sustained production of cytokines made by a distinct lineage of Th cells, termed Th17 cells. The demonstration that Th17-related cytokines cause pathology in many organs, including the gut, and that expansion and maintenance of Th17 cell responses require the activity of IL-23, a cytokine made in excess in the gut of IBD patients has contributed to elucidate new pathways of intestinal tissue damage as well as to design new therapeutic strategies. In this review, we discuss the available data supporting the role of the IL-23/Th17 axis in the modulation of intestinal tissue inflammation.
Salvage of Upper Limb following a Severe Crushing Trauma: Immediate Reconstruction with a Free Flap and Subsequent Hyperbaric Oxygen Therapy
Authors: Serra M. P. , Longhi P.
Published: May 2009

A microsurgical latissimus dorsi flap was performed for resurfacing a large soft tissue defect of the forearm with exposure of the vital structures and contaminated wound. Early coverage of a defect is a generally accepted concept to achieve a better functional result. The authors present a case report where a free latissimus dorsi flap with subsequent hyperbaric oxygen therapy allowed a successful single stage reconstruction of this complex severely contaminated defect.
GCFR Coupled Neutronic and Thermal‐Fluid‐Dynamics Analyses for a Core Containing Minor Actinides
Authors: Castelliti Diego , Bomboni Eleonora , Cerullo Nicola , Lomonaco Guglielmo , Parisi Carlo
Published: May 2009

Problems about future energy availability, climate changes, and air quality seem to play an important role in energy production. While current reactor generations provide a guaranteed and economical energy production, new nuclear power plant generation would increase the ways and purposes in which nuclear energy can be used. To explore these new technological applications, several governments, industries, and research communities decided to contribute to the next reactor generation, called “Generation IV.” Among the six Gen‐IV reactor designs, the Gas Cooled Fast Reactor (GCFR) uses a direct‐cycle helium turbine for electricity generation and for a CO2‐free thermochemical production of hydrogen. Additionally, the use of a fast spectrum allows actinides transmutation, minimizing the production of long‐lived radioactive waste in an integrated fuel cycle. This paper presents an analysis of GCFR fuel cycle optimization and of a thermal‐hydraulic of a GCFR‐prototype under steady‐state and transient conditions. The fuel cycle optimization was performed to assess the capability of the GCFR to transmute MAs, while the thermal‐hydraulic analysis was performed to investigate the reactor and the safety systems behavior during a LOFA. Preliminary results show that limited quantities of MA are not affecting significantly the thermal‐fluid‐dynamics behavior of a GCFR core.
Critical Role for Caspase-8 in Epidermal Growth Factor Signaling
Authors: Finlay Darren , Howes Amy , Vuori Kristiina
Published: May 2009

Caspase-8 has a well-defined canonical role as an apical protease of the extrinsic apoptosis pathway. Evidence is growing, however, that the protein has numerous other nonapoptotic functions. We have previously shown that caspase-8 is required for efficient adhesion-induced activation of the extracellular signal–regulated kinase (Erk)-1/2 pathway. We now show that caspase-8 is also necessary for the efficient activation of downstream events associated with epidermal growth factor (EGF) signaling. This promotion of EGF-induced Erk1/2 activation is independent of the proteolytic activity of caspase-8 and can be recapitulated using only the pro-domains of the protein. In addition, we identify specific residues within the caspase-8 “RXDLL motif” that are essential for Erk pathway activation. Furthermore, these residues are also involved in forming a complex with the tyrosine kinase Src. Caspase-8 null cells and cells reconstituted with caspase-8 harboring point mutations of these critical amino acids also show defective EGF-induced migration as compared with cells reconstituted with the wild-type protein. In sum, we provide the first evidence for caspase-8 as an essential component of growth factor signaling and suggest that this may be due to its association with Src. As the EGF/Src pathway activity has been shown to promote oncogenic events, our findings that caspase-8 is necessary for these activities may help explain why it is rarely deleted or silenced in tumors. [Cancer Res 2009;69(12):5023–9]
Patterns of Gene Expression and Copy-Number Alterations in von-Hippel Lindau Disease-Associated and Sporadic Clear Cell Carcinoma of the Kidney
Authors: Beroukhim Rameen , Brunet Jean-Philippe , Di Napoli Arianna , Mertz Kirsten D. , Seeley Apryle , Pires Maira M. , Linhart David , Worrell Robert A. , Moch Holger , Rubin Mark A. , Sellers William R. , Meyerson Matthew , Linehan W. Marston , Kaelin William G. , Signoretti Sabina
Published: May 2009

Recent insights into the role of the von-Hippel Lindau (VHL) tumor suppressor gene in hereditary and sporadic clear-cell renal cell carcinoma (ccRCC) have led to new treatments for patients with metastatic ccRCC, although virtually all patients eventually succumb to the disease. We performed an integrated, genome-wide analysis of copy-number changes and gene expression profiles in 90 tumors, including both sporadic and VHL disease-associated tumors, in hopes of identifying new therapeutic targets in ccRCC. We identified 14 regions of nonrandom copy-number change, including 7 regions of amplification (1q, 2q, 5q, 7q, 8q, 12p, and 20q) and 7 regions of deletion (1p, 3p, 4q, 6q, 8p, 9p, and 14q). An analysis aimed at identifying the relevant genes revealed VHL as one of three genes in the 3p deletion peak, CDKN2A and CDKN2B as the only genes in the 9p deletion peak, and MYC as the only gene in the 8q amplification peak. An integrated analysis to identify genes in amplification peaks that are consistently overexpressed among amplified samples confirmed MYC as a potential target of 8q amplification and identified candidate oncogenes in the other regions. A comparison of genomic profiles revealed that VHL disease-associated tumors are similar to a subgroup of sporadic tumors and thus more homogeneous overall. Sporadic tumors without evidence of biallelic VHL inactivation fell into two groups: one group with genomic profiles highly dissimilar to the majority of ccRCC and a second group with genomic profiles that are much more similar to tumors with biallelic inactivation of VHL. [Cancer Res 2009;69(11):4674–81]
Extranuclear Coactivator Signaling Confers Insensitivity to Tamoxifen
Authors: Kumar Rakesh , Zhang Hao , Holm Caroline , Vadlamudi Ratna K. , Landberg Goran , Rayala Suresh K.
Published: May 2009

Purpose: Tamoxifen is one of many standard therapeutic options currently available for estrogen receptor-α–positive breast cancer patients. Emerging data have suggested that levels of estrogen receptor coregulatory proteins play a significant role in acquiring resistance to antiestrogen action. It has been suggested that high levels of estrogen receptor coactivators and its mislocalization may enhance the estrogen agonist activity of tamoxifen and contribute to tamoxifen resistance.

Experimental Design: In an effort to understand the impact of nongenomic signaling and its contribution to hormone resistance in a whole-animal setting, we generated a transgenic mouse expressing a cytoplasmic version of proline-, glutamic acid–, and leucine-rich protein–1 (PELP1) mutant defective in its nuclear translocation (PELP1-cyto) and implanted these mice with tamoxifen pellets to assess its responsiveness.

Results: We show that mammary glands from these mice developed widespread hyperplasia with increased cell proliferation and enhanced activation of mitogen-activated protein kinase and AKT as early as 12 weeks of age. Treatment with tamoxifen did not inhibit this hyperplasia; instead, such treatment exaggerated hyperplasia with an enhanced degree of alteration, indicative of hypersensitivity to tamoxifen. Analysis of molecular markers in the transgenic mammary glands from the tamoxifen-treated transgenic mice showed higher levels of proliferation markers proliferating cell nuclear antigen and activated mitogen-activated protein kinase than in untreated PELP1-cyto cell-derived mice. We also found that nude mice with MCF-7/PELP1-cyto cell-derived tumor xenografts did not respond to tamoxifen. Using immunohistochemical analysis, we found that 43% of human breast tumor samples had high levels of cytoplasmic PELP1, which shows a positive correlation between tumor grade and proliferation. Patients whose tumors had high levels of cytoplasmic PELP1 exhibited a tendency to respond poorly to tamoxifen compared with patients whose tumors had low levels of cytoplasmic PELP1.

Conclusions: These findings suggest that PELP1 localization could be used as a determinant of hormone sensitivity or vulnerability. The establishment of the PELP1-cyto transgenic mouse model is expected to facilitate the development of preclinical approaches for effective intervention of breast tumors using cytoplasmic coregulators and active nongenomic signaling.
Therapeutic Efficacy of Seliciclib in Combination with Ionizing Radiation for Human Nasopharyngeal Carcinoma
Authors: Hui Angela B.Y. , Yue Shijun , Shi Wei , Alajez Nehad M. , Ito Emma , Green Simon R. , Frame Sheelagh , O'Sullivan Brian , Liu Fei-Fei
Published: May 2009

Purpose: Seliciclib is a small-molecule cyclin-dependent kinase inhibitor, which has been reported to induce apoptosis and cell cycle arrest in EBV-negative nasopharyngeal carcinoma cell lines. Because most nasopharyngeal carcinoma patients harbor EBV, we proceeded to evaluate the cytotoxic effects of seliciclib in EBV-positive nasopharyngeal carcinoma models.

Experimental Design: Cytotoxicity of seliciclib was investigated in the EBV-positive cell line C666-1 and the C666-1 and C15 xenograft models. Caspase activities and cell cycle analyses were measured by flow cytometry. Efficacy of combined treatment of seliciclib with radiation therapy was also evaluated.

Results: Seliciclib caused significant cytotoxicity in the C666-1 cells in a time- and dose-dependent manner, with accumulation of cells in both sub-G1 and G2-M phases, indicative of apoptosis and cell cycle arrest, respectively. Caspase-2, -3, -8, and -9 activities were all increased, with caspase-3 being the most significantly activated at 48 h after treatment. These cells also showed a reduction of Mcl-1 mRNA and protein levels. Combined treatment of seliciclib with radiation therapy showed a synergistic interaction with enhanced cytotoxicity in C666-1 cells and delayed repair of double-strand DNA breaks. For in vivo models, significant delays in tumor growth were observed for both C666-1 and C15 tumors, which were associated with enhanced apoptosis as determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and immunohistochemistry analyses.

Conclusions: Seliciclib enhanced the antitumor efficacy of radiation therapy in EBV-positive nasopharyngeal carcinoma, characterized by G2-M arrest, and apoptosis, associated with an induction in caspase activity. This process is mediated by reduction in Mcl-1 expression and by attenuation of double-strand DNA break repair.
Activation of the Unfolded Protein Response Is Associated with Favorable Prognosis in Acute Myeloid Leukemia
Authors: Schardt Julian A. , Weber Daniel , Eyholzer Marianne , Mueller Beatrice U. , Pabst Thomas
Published: May 2009

Purpose: The unfolded protein response is triggered by the accumulation of misfolded proteins within the endoplasmic reticulum. Previous studies suggest that the unfolded protein response is activated in some cancer cell lines and involved in tumor development. The role of the unfolded protein response during leukemogenesis is unknown thus far.

Experimental Design: Here, we assessed the induction of key effectors of the unfolded protein response in leukemic cells at diagnosis of 105 acute myeloid leukemia (AML) patients comprising all subtypes. We determined the formation of the spliced variant of the X-box–binding protein 1 (XBP1) mRNA, as well as expression levels of calreticulin, GRP78, and CHOP mRNA.

Results: The formation of the spliced variant of XBP1s was detectable in 16.2% (17 of 105) of AML patients. Consistent with activated unfolded protein response, this group also had significantly increased expression of calreticulin, GRP78, and CHOP. AML patients with activated unfolded protein response had lower WBC counts, lactate dehydrogenase levels, and more frequently, secondary AML. The incidence of fms-related tyrosine kinase 3 (FLT3) mutations was significantly lower in patients with activated unfolded protein response. In addition, an association was observed between activated unfolded protein response and deletion of chromosome 7. Finally, the clinical course of AML patients with activated unfolded protein response was more favorable with lower relapse rate (P = 0.0182) and better overall (P = 0.041) and disease-free survival (P = 0.022).

Conclusions: These results suggest that the unfolded protein response is activated in a considerable subset of AML patients. AML patients with activated unfolded protein response present specific clinical characteristics and a more favorable course of the disease.
Imaging a Genetically Engineered Oncolytic Vaccinia Virus (GLV-1h99) Using a Human Norepinephrine Transporter Reporter Gene
Authors: Brader Peter , Kelly Kaitlyn J. , Chen Nanhai , Yu Yong A. , Zhang Qian , Zanzonico Pat , Burnazi Eva M. , Ghani Rashid E. , Serganova Inna , Hricak Hedvig , Szalay Aladar A. , Fong Yuman , Blasberg Ronald G.
Published: May 2009

Purpose: Oncolytic viral therapy continues to be investigated for the treatment of cancer, and future studies in patients would benefit greatly from a noninvasive modality for assessing virus dissemination, targeting, and persistence. The purpose of this study was to determine if a genetically modified vaccinia virus, GLV-1h99, containing a human norepinephrine transporter (hNET) reporter gene, could be sequentially monitored by [123I]metaiodobenzylguanidine (MIBG) γ-camera and [124I]MIBG positron emission tomography (PET) imaging.

Experimental Design: GLV-1h99 was tested in human malignant mesothelioma and pancreatic cancer cell lines for cytotoxicity, expression of the hNET protein using immunoblot analysis, and [123I]MIBG uptake in cell culture assays. In vivo [123I]MIBG γ-camera and serial [124I]MIBG PET imaging was done in MSTO-211H orthotopic pleural mesothelioma tumors.

Results: GLV-1h99 successfully infected and provided dose-dependent levels of transgene hNET expression in human malignant mesothelioma and pancreatic cancer cells. The time course of [123I]MIBG accumulation showed a peak of radiotracer uptake at 48 hours after virus infection in vitro. In vivo hNET expression in MSTO-211H pleural tumors could be imaged by [123I]MIBG scintigraphy and [124I]MIBG PET 48 and 72 hours after GLV-1h99 virus administration. Histologic analysis confirmed the presence of GLV-1h99 in tumors.

Conclusion: GLV-1h99 shows high mesothelioma tumor cell infectivity and cytotoxic efficacy. The feasibility of imaging virus-targeted tumor using the hNET reporter system with [123I]MIBG γ-camera and [124I]MIBG PET was shown in an orthotopic pleural mesothelioma tumor model. The inclusion of human reporter genes into recombinant oncolytic viruses enhances the potential for translation to clinical monitoring of oncolytic viral therapy.
HGF and c-Met Participate in Paracrine Tumorigenic Pathways in Head and Neck Squamous Cell Cancer
Authors: Knowles Lynn M. , Stabile Laura P. , Egloff Ann Marie , Rothstein Mary E. , Thomas Sufi M. , Gubish Christopher T. , Lerner Edwina C. , Seethala Raja R. , Suzuki Shinsuke , Quesnelle Kelly M. , Morgan Sarah , Ferris Robert L. , Grandis Jennifer R. , Siegfried Jill M.
Published: May 2009

Purpose: We determined hepatocyte growth factor (HGF) and c-Met expression and signaling in human head and neck squamous cell carcinoma (HNSCC) cells and primary tissues and tested the ability of c-Met tyrosine kinase inhibitors (TKI) to block HGF-induced biological signaling.

Experimental Design: Expression and signaling were determined using immunoblotting, ELISA, and immunohistochemistry. Biological end points included wound healing, cell proliferation, and invasion. c-Met TKIs were tested for their ability to block HGF-induced signaling and biological effects in vitro and in xenografts established in nude mice.

Results: c-Met was expressed and functional in HNSCC cells. HGF was secreted by HNSCC tumor-derived fibroblasts, but not by HNSCC cells. Activation of c-Met promoted phosphorylation of AKT and mitogen-activated protein kinase as well as release of the inflammatory cytokine interleukin-8. Cell growth and wound healing were also stimulated by HGF. c-Met TKIs blocked HGF-induced signaling, interleukin-8 release, and wound healing. Enhanced invasion of HNSCC cells induced by the presence of tumor-derived fibroblasts was completely blocked with a HGF-neutralizing antibody. PF-2341066, a c-Met TKI, caused a 50% inhibition of HNSCC tumor growth in vivo with decreased proliferation and increased apoptosis within the tumors. In HNSCC tumor tissues, both HGF and c-Met protein were increased compared with expression in normal mucosa.

Conclusions: These results show that HGF acts mainly as a paracrine factor in HNSCC cells, the HGF/c-Met pathway is frequently up-regulated and functional in HNSCC, and a clinically relevant c-Met TKI shows antitumor activity in vivo. Blocking the HGF/c-Met pathway may be clinically useful for the treatment of HNSCC.
Phenylmethimazole Decreases Toll-Like Receptor 3 and Noncanonical Wnt5a Expression in Pancreatic Cancer and Melanoma Together with Tumor Cell Growth and Migration
Authors: Schwartz Anthony L. , Malgor Ramiro , Dickerson Eric , Weeraratna Ashani T. , Slominski Andrzej , Wortsman Jacobo , Harii Norikazu , Kohn Aimee D. , Moon Randall T. , Schwartz Frank L. , Goetz Douglas J. , Kohn Leonard D. , McCall Kelly D.
Published: May 2009

Purpose: To evaluate whether (a) Wnt5a expression in pancreatic cancer and malignant melanoma cells might be associated with constitutive levels of Toll-like receptor 3 (TLR3) and/or TLR3 signaling; (b) phenylmethimazole (C10), a novel TLR signaling inhibitor, could decrease constitutive Wnt5a and TLR3 levels together with cell growth and migration; and (c) the efficacy of C10 as a potential inhibitor of pancreatic cancer and malignant melanoma cell growth in vivo.

Experimental Design: We used a variety of molecular biology techniques including but not limited to PCR, Western blotting, and ELISA to evaluate the presence of constitutively activated TLR3/Wnt5a expression and signaling. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide-based technology and scratch assays were used to evaluate inhibition of cell growth and migration, respectively. TLR3 regulation of cell growth was confirmed using small interfering RNA technology. Nude and severe combined immunodeficient mice were implanted with human pancreatic cancer and/or melanoma cells and the effects of C10 on tumor growth were evaluated.

Results: We show that constitutive TLR3 expression is associated with constitutive Wnt5a in human pancreatic cancer and malignant melanoma cell lines, that C10 can decrease constitutive TLR3/Wnt5a expression and signaling, suggesting that they are interrelated signal systems, and that C10 inhibits growth and migration in both of these cancer cell lines. We also report that C10 is effective at inhibiting human pancreatic cancer and malignant melanoma tumor growth in vivo in nude or severe combined immunodeficient mice and associate this with inhibition of signal transducers and activators of transcription 3 activation.

Conclusions: C10 may have potential therapeutic applicability in pancreatic cancer and malignant melanoma.
Association between Plasma 25-Hydroxyvitamin D and Breast Cancer Risk
Authors: Crew Katherine D. , Gammon Marilie D. , Steck Susan E. , Hershman Dawn L. , Cremers Serge , Dworakowski Elzbieta , Shane Elizabeth , Terry Mary Beth , Desai Manisha , Teitelbaum Susan L. , Neugut Alfred I. , Santella Regina M.
Published: May 2009

Vitamin D has been associated with decreased risk of several cancers. In experimental studies, vitamin D has been shown to inhibit cell proliferation and induce differentiation and apoptosis in normal and malignant breast cells. Using a population-based case-control study on Long Island, New York, we examined the association of breast cancer with plasma 25-hydroxyvitamin D (25-OHD) levels, a measure of vitamin D body stores. In-person interviews and blood specimens were obtained from 1,026 incident breast cancer cases diagnosed in 1996 to 1997 and 1,075 population-based controls. Plasma 25-OHD was measured in batched, archived specimens by Diasorin RIA. The mean (SD) plasma 25-OHD concentration was 27.1 (13.0) and 29.7 (15.1) ng/mL in the cases and controls, respectively (P < 0.0001). Plasma 25-OHD was inversely associated with breast cancer risk in a concentration-dependent fashion (Ptrend = 0.002). Compared with women with vitamin D deficiency (25-OHD, <20 ng/mL), levels above 40 ng/mL were associated with decreased breast cancer risk (odds ratio, 0.56; 95% confidence interval, 0.41-0.78). The reduction in risk was greater among postmenopausal women (odds ratio, 0.46; 95% confidence interval, 0.09-0.83), and the effect did not vary according to tumor hormone receptor status. In summary, these results add to a growing body of evidence that adequate vitamin D stores may prevent breast cancer development. Whereas circulating 25-OHD levels of >32 ng/mL are associated with normal bone mineral metabolism, our data suggest that the optimal level for breast cancer prevention is ≥40 ng/mL. Well-designed clinical trials are urgently needed to determine whether vitamin D supplementation is effective for breast cancer chemoprevention.
Lung Cancer Inhibitory Effect of Epigallocatechin-3-Gallate Is Dependent on Its Presence in a Complex Mixture (Polyphenon E)
Authors: Fu Huijing , He Jun , Mei Fan , Zhang Qi , Hara Yukihiko , Ryota Seto , Lubet Ronald A. , Chen Ruth , Chen Da-Ren , You Ming
Published: May 2009

Green tea has been shown to exhibit cancer-preventive activities in preclinical studies. However, (−)-epigallocatechin-3-gallate (EGCG) alone was shown to be ineffective in preventing lung tumorigenesis in mice by aerosol administration. In this study, Polyphenon E and Polyphenon E without EGCG were administered by aerosol delivery to A/J mice 2 weeks after carcinogen treatment and continuing daily throughout the remainder of the study (20 weeks). An improved aerosol delivery system with a custom-built atomizer, an efficient solvent remove system, and a nose-only exposure chamber was used to provide aerosols with stable size distribution. There were no significant differences in the size distributions of Polyphenon E and Polyphenon E without EGCG. With a relatively low dose level (4.19 mg/kg), Polyphenon E decreased tumor multiplicity by 53%, whereas Polyphenon E without EGCG at the same dose failed to inhibit lung carcinogenesis. These results indicate that aerosol administration can be an effective approach in chemoprevention study, and aerosolized Polyphenon E can significantly inhibit pulmonary adenoma formation and growth in A/J mice. Furthermore, in aerosolized form, EGCG, which is thought to be the most active component of Polyphenon E, has to be present with other tea catechins to show chemopreventive activity on lung tumorigenesis.
Conjunctiva-Associated Lymphoid Tissue – Current Knowledge, Animal Models and Experimental Prospects
Authors: Steven Philipp , Gebert Andreas
Published: May 2009

The conjunctiva contains organized lymphoid tissue that consists of intraepithelial lymphocytes, subepithelial lymphoid follicles and adjacent lymphatics and blood vessels. This conjunctiva-associated lymphoid tissue (CALT) is assumed to play a key role in the protection of the ocular surface by initiating and regulating immune responses. The article reviews the current knowledge of CALT in humans and compares its structure and functions with mucosa-associated lymphoid tissues at other mucosal sites. An update is given on CALT animal models and on functional studies on the transepithelial transport of microorganisms and antigenic substances by specialized epithelial cells. The use of two-photon microscopy is reviewed regarding its importance to the understanding of dynamic processes going on within CALT.
Electron Impact Mass Spectroscopic Studies on Mouse Retinal Fatty Acids
Authors: Hegde K.R. , Varma S.D.
Published: May 2009

Aim: The primary objective of these investigations was to determine the fatty acid composition of the mouse retina as affected by diabetes. Additionally, in order to ascertain if there is any accumulation of lipids in the diabetic retina as occurs in many diabetic tissues, its total fatty acid content was also determined. Methods: Lipids in the retina of normal and diabetic mice were trans-methylated with methanolic HCl. The esters so prepared were analyzed for fatty acids by gas chromatography-mass spectrometry, qualitatively as well as quantitatively. Results: The major fatty acids in the retina were palmitic (PA), oleic (OA), stearic (SA), arachidonic (AA) and docosahexaenoic (DHA) acids. The content of all these fatty acids increased significantly in the diabetic retina, reflecting lipidosis. The major increases (∼3 times the normal) were found in PA, OA, SA and AA. The increase in DHA, however, was much less (∼1.4 times). The relative percentages of fatty acids were also affected. While the relative percentages of PA and OA increased in the diabetic retina, there were insignificant changes in the percentages of SA and AA. Interestingly, the relative percentage of DHA underwent a significant decrease, about 50% of the normal. Conclusions: The results show that there is excessive accumulation of lipids in the diabetic retina. This is consistent with the known increased mobilization of lipids from the adipose tissue and their accumulation in other tissues under diabetic conditions. On a percentage profile basis, while the relative percentage of most of the fatty acids increased, the DHA percentage significantly decreased. This could be explained by its dilution by the fatty acids coming from the fat depots which lack DHA. Physiologically, the excessive accumulation of fatty acids in the diabetic retina correlates with the lipofuscinosis and neural dysfunction associated with this disease.
Octreotide Inhibits Choroidal Neovascularization in Rats
Authors: Qu Yi , Zhang Shuihua , Xu Xiaoyi , Wang Hong , Li Jinghai , Zhou Fang , Wei Fengcai
Published: May 2009

Aims: Octreotide exhibits anti-angiogenic activity in animal models of retinopathy of prematurity and in clinical cases of proliferative diabetic retinopathy. In this study, we tested the applicability of using octreotide for inhibiting experimental choroidal neovascularization (CNV) in rats. Methods: Of 15 adult rats used, 3 served as non-laser-treated controls. CNV was induced in the right eye of the remaining 12 rats by laser photocoagulation. These 12 rats were divided into two groups (n = 6 each) which were retrobulbarly injected with octreotide (50 μg/kg) or phosphate-buffered saline (PBS) (0.15 ml) immediately and on day 3 after the first injection. Fluorescein angiography and quantitative analysis of the retinal pigment epithelium (RPE)-choroidal flat mounts were performed 14 days after the operation to evaluate the changes in the CNV lesions. Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) was used to compare the changes shown by the octreotide-, PBS-, and non-laser-treated rats (n = 3 each) with regard to mRNA levels of the vascular endothelial growth factor (VEGF) and insulin-like growth factor (IGF)-1 in the RPE-choroidal complex. Results: Octreotide treatment significantly inhibited fluorescein leakage and the extent of neovascularization as was observed in the flat mounts of choroidal tissue derived from the rats with induced CNV. The VEGF and IGF-1 mRNA levels were reduced following treatment with octreotide. Conclusion: The retrobulbar administration of octreotide may be an effective new therapeutic strategy for CNV.