Message Level Research Articles

Role of slug in TGF-β1 mediated MMP-9 expression in human oral squamous cell cancer

21000 Background: Oral squamous cell cancer (OSCC) causes significant morbidity and mortality as a result of loco-regional invasion and distant metastasis. Invasive OSCC tumors demonstrate increased expression of matrix metalloproteinases (MMPs), including MMP-9. TGF-β1, which promotes MMP-dependent invasion, is also frequently overexpressed in OSCC. Recent reports indicate that TGF-β1 can signal through Snail family of transcription factors, including Slug, which are key regulators of epithelial to mesenchymal transition. Methods: To examine the relationship between TGF-β1 and MMP-9 in human OSCC tumors, phospho-Smad2 and MMP-9 expression was quantified in an oral cancer tissue microarray, and then the relationship determined using Pearson correlation coefficient. To understand the role of Slug in TGF-β1-mediated MMP-9 expression, premalignant oral keratinocyte cell lines (OKF4 and OKF6) and malignant OSCC lines (SCC25 and UMSCC1) were treated with TGF-β1 (10 ng/ml) and analyzed for Slug and MMP-9 expression at the message and protein levels. The specific role of Slug was determined by transfecting OSCC cells with Slug specific siRNA or control siRNA, treating with TGF-β1 for 24h, and determining the effect on MMP-9 expression. Results: There was a statistically significant correlation between phospho-Smad2 and MMP- 9 expression in human OSCC samples, with a Pearson correlation coefficient of 0.4392 (p=0.0007). In vitro, treatment of premalignant and OSCC cells with TGF-β1 induced both Slug and MMP-9 expression at the message and protein level. Interestingly, transfection of Slug siRNA in OKF4 and OKF6 cells increased both basal and TGF-β1-mediated MMP-9 expression, while transfection of Slug siRNA in SCC25 and UMSCC1 cells decreased basal and TGF-β1-mediated MMP-9 expression. Conclusions: Overall, our data indicate that TGF-β1 may regulate MMP-9 expression in human oral squamous cell cancer. Additionally, our data indicate a role for Slug in TGF-β1-mediated MMP-9 expression in oral cancer cells. Future experiments will examine the mechanism by which Slug differentially regulates MMP-9 expression in premalignant and malignant OSCC cells. No significant financial relationships to disclose.

Apyrases (Nucleoside Triphosphate-Diphosphohydrolases) Play a Key Role in Growth Control in Arabidopsis

Expression of two Arabidopsis (Arabidopsis thaliana) apyrase (nucleoside triphosphate-diphosphohydrolase) genes with high similarity, APY1 and APY2, was analyzed during seedling development and under different light treatments using beta-glucuronidase fusion constructs with the promoters of both genes. As evaluated by beta-glucuronidase staining and independently confirmed by other methods, the highest expression of both apyrases was in rapidly growing tissues and/or tissues that accumulate high auxin levels. Red-light treatment of etiolated seedlings suppressed the protein and message level of both apyrases at least as rapidly as it inhibited hypocotyl growth. Adult apy1 and apy2 single mutants had near-normal growth, but apy1apy2 double-knockout plants were dwarf, due primarily to reduced cell elongation. Pollen tubes and etiolated hypocotyls overexpressing an apyrase had faster growth rates than wild-type plants. Growing pollen tubes released ATP into the growth medium and suppression of apyrase activity by antiapyrase antibodies or by inhibitors simultaneously increased medium ATP levels and inhibited pollen tube growth. These results imply that APY1 and APY2, like their homologs in animals, act to reduce the concentration of extracellular nucleotides, and that this function is important for the regulation of growth in Arabidopsis.

Biosynthesis of Wdr16, a marker protein for kinocilia‐bearing cells, starts at the time of kinocilia formation in rat, andwdr16gene knockdown causes hydrocephalus in zebrafish1

The rat ortholog of the WD40 repeat protein Wdr16 is abundantly expressed in testis and cultured ependymal cells. Low levels are found in lung and brain, respectively, while it is absent from kinocilia-free tissues. In testis and ependymal primary cultures, Wdr16 messenger RNA appears concomitantly with the messages for sperm-associated antigen 6, a kinocilia marker, and for hydin, a protein linked to ciliary function and hydrocephalus. In testis, ependyma and respiratory epithelium, the Wdr16 protein is up-regulated together with kinocilia formation. The wdr16 gene is restricted to genera in possession of kinocilia, and it is strongly conserved during evolution. The human and zebrafish proteins are identical in 62% of their aligned amino acids. On the message level, the zebrafish Wdr16 ortholog was found exclusively in kinocilia-bearing tissues by in situ hybridisation. Gene knockdown in zebrafish embryos by antisense morpholino injection resulted in severe hydrocephalus formation with unaltered ependymal morphology or ciliary beat. Wdr16 can be considered a differentiation marker of kinocilia-bearing cells. In the brain, it appears to be functionally related to water homeostasis or osmoregulation.

Stress response of genes encoding putative stress signaling molecules of Mycobacterium tuberculosis

Mycobacterium tuberculosis possesses six genes (Rv0516c, Rv1364c, Rv1365c, Rv1904, Rv2638 and Rv3687c) encoding putative anti-sigma factor antagonists or stress signaling molecules (SSMs). We have previously shown that the products of these genes physically interact between themselves and with sigma factor SigF (encoded by Rv3286c) and anti-sigma factor RsbW (encoded by Rv3287c) in the yeast two-hybrid system. In order to understand whether ssms respond to stress, we analyzed the expression of these genes in M. tuberculosis exposed to stress at message level using real time RT-PCR. The results revealed that most ssms of M. tuberculosis responded to stress and Rv0516c was the most prominent one. Rv0516c showed elevated expression for NaCl, oxidative and starvation stresses and this was followed by Rv2638 which exhibited upregulation towards stationary phase, heat and oxidative stresses. While Rv1904 and Rv3687c responded significantly to cold and oxidative stresses, Rv1364c responded only to heat stress. Further, studies on the response of sigF and rsbW to stress revealed that only rsbW significantly responded to heat, cold, oxidative, starvation and anaerobic stresses. The response of ssms and rsbW to different stresses may be an indication for the stress activation and regulation of SigF by these molecules.

A message-level security model consisting of multiple security-tokens mechanism

Current Web services architectures are confronted with a few stubborn problems, and the security problem becomes one of the bottlenecks that restrict the extensive application of Web service. After compared transport level with message level security, the limitation of transport level security became clearly. And then on the basis of the analysis message level security, especially WS-Security, XML security protocol was adopted to guarantee message level security. Because WS-Security is a new protocol jointly developed by Microsoft and IBM among these XML security protocols, and a security-token mechanism of WS-Security mechanism was improved, added a table with security-token types. And a new model consisting of multiple security-token is put forward to guarantee the security of message transmission.

Temporal change in gene expression in the rat dentate gyrus following passive avoidance learning

A learning event initiates a cascade of altered gene expression leading to synaptic remodelling within the hippocampal dentate gyrus, a structure vital to memory formation. To illuminate this transcriptional program of synaptic plasticity we used microarrays to quantify mRNA from the rat dentate gyrus at increasing times following passive avoidance learning. Approximately, 500 known genes were transcriptionally regulated across the 24 h post-training period. The 0-2 h period saw up-regulation of genes involved in transcription while genes with a role in synaptic/cytoskeletal structure increased 0-6 h, consistent with structural rearrangements known to occur at these times. The most striking feature was the profound down-regulation, across all functional groups, 12 h post-training. Bioinformatics analysis identified the likely transcription factors controlling gene expression in each post-training period. The role of NF kappa B, implicated in the early post-training period was subsequently confirmed with activation and nuclear translocation seen in dentate granule neurons following training. mRNA changes for four genes, LRP3 (0 h), alpha actin (3 h), SNAP25 and NSF (6-12 h), were validated at message and/or protein level and shown to be learning specific. Thus, the memory-associated transcriptional cascade supports the cardinal periods of synaptic loosening, reorganisation and selection thought to underpin the process of long-term memory consolidation in the hippocampus.

Upregulation of AKT1 protein expression in forskolin‐stimulated macrophage: Evidence from ChIP analysis that CREB binds to and activates the AKT1 promoter

Recently, we reported that silencing CREB gene expression by RNAi significantly attenuates forskolin-induced activation of Akt1. We now provide evidence that forskolin-treatment causes transcriptional and translational upregulation of Akt1 in macrophages. Akt synthesis was demonstrated by [(14)C]leucine or [(35)S] incorporation into newly synthesized Akt1 protein. Akt protein levels increased by approximately 1.5-fold after only a 5 min exposure of macrophages to forskolin. Akt1 levels thereafter rapidly returned to basal values (t(1/2) approximately 15 min). Maximal upregulation of Akt1 occurred in cells treated with 10 microM forskolin. Forskolin-dependent Akt1 synthesis was abolished by pretreating the cells with CREB-directed dsRNA as demonstrated at both the message and protein level, as well as by determining the synthesis of [(35)S]-labeled Akt1 protein. The PKA inhibitor H-89, greatly attenuated forskolin-induced Akt1 synthesis. Transcriptional and translational inhibitors also greatly reduced Akt1 synthesis in forskolin-stimulated [(14)C]leucine-labeled macrophages. Using a chromatin immunoprecipitation assay, we demonstrate that CREB binds to a CRE binding domain of the Akt1 gene promoter. In conclusion, we show here for the first time transcriptional upregulation of Akt1 by CREB, based upon Akt1 protein synthesis and its modulation by transitional and translational inhibitors in forskolin-stimulated cells, Akt1 protein. and mRNA levels upon silencing CREB gene expression, and binding of CREB to the Akt1 gene promoter.

Online discussion processes: Effects of earlier messages’ evaluations, knowledge content, social cues and personal information on later messages

This study of the flow of online discussions examined how earlier messages affected later messages along five dimensions: (1) evaluations (agreement, disagreement, or unresponsive actions); (2) knowledge content (contribution, repetition, or null content); (3) social cues (positive, negative, or none); (4) personal information (number of visits); and (5) elicitation (eliciting response or not). Using sequential logit regressions and a structural equation model (SEM), this study analyzed 131 messages across seven topics in the mathematics forum of a university Bulletin Board System (BBS) Website. Results showed that a disagreement or contribution in the previous message increased the likelihoods of disagreements and social cue displays in the current message. Unlike face-to-face discussions, online discussion messages that disagreed with an earlier message were more likely to elicit responses. Together, these results support the claims that teachers can use and manage online discussions at the message level to promote critical thinking, facilitate discussion of controversial topics, and reduce status effects.

Structurally well-formed narrative production in the face of severe conceptual deterioration: A longitudinal case study of a woman with semantic dementia

Language production was examined in a woman with semantic dementia (SD) over a three-year time period. Narratives were analyzed for their propositional content, syntactic structure, morphological complexity, and lexical makeup. An additional analysis looked at the proportion of vague terms and pronouns out of all nominal expressions. Investigation of verb frequency, light/heavy verbs, verb type/token ratio, and verb argument structure was also conducted. Findings suggest that alongside a severe deterioration of the general message level and continual degradation of lexical retrieval mechanisms, syntactic and morphological aspects of connected speech remain essentially unaffected by SD. It is argued that SD leads to a dissociation between impaired conceptual knowledge and intact structure in language production.

Channel catfish, Ictalurus punctatus, CD4-like molecules

Two CD4-like (CD4L) molecules have been identified in channel catfish, Ictalurus punctatus. Although phylogenetically related to other vertebrate CD4 molecules, they exhibit only 19% amino acid identity to each other. IpCD4L-1 encodes a predicted protein containing four immunoglobulin domains, a transmembrane region and a cytoplasmic tail containing a p56 lck binding site. In contrast, IpCD4L-2 encodes for a similar protein with three immunoglobulin domains. The gene organization of IpCD4L-1 is very similar to that of other vertebrate CD4 genes, while the genomic organization of IpCD4L-2 is different. Southern blots indicate both catfish molecules are likely single copy genes and mapping studies show that both are found on a single Bacterial Artificial Chromosome suggesting close linkage. At the message level, IpCD4L-1 and -2 are expressed in various catfish lymphoid tissues and in non-B-cell peripheral blood leukocytes (PBL). Both messages are upregulated in concanavalin A (ConA) and alloantigen stimulated PBL, but not in lipopolysaccharide (LPS)-stimulated cultures. In contrast, they are differentially expressed among the catfish clonal T cell lines. While both molecules appear to be T cell specific, their functional significance in catfish is unknown.

Semaphorin 4D provides a link between axon guidance processes and tumor-induced angiogenesis

Tumor progression and metastasis depend on the ability of cancer cells to initiate angiogenesis and ensure delivery of oxygen, nutrients, and growth factors to rapidly dividing transformed cells and provide access to the systemic circulation. In addition to well established growth factors and inflammatory mediators that promote capillary sprouting and endothelial cell growth and migration, an emerging body of evidence supports a previously unrecognized function for axon guidance molecules in regulation of blood vessel development. Here we show that semaphorin 4D (Sema4D), a protein originally shown to regulate axonal growth cone guidance in the developing central nervous system through its receptor, plexin-B1, is highly expressed in cell lines derived from head and neck squamous cell carcinomas (HNSCCs) at both the protein and message level. Immunohistochemical analysis of a large collection of HNSCC specimens revealed high levels of Sema4D in a cell surface pattern in invading islands of transformed epithelial cells, but not in normal and noninvasive dysplastic epithelium. A similar pattern was observed in malignant cells from prostate, colon, breast, and lung cancer tissues. When shed from HNSCC cells, Sema4D stimulates endothelial cell migration, which can be prevented by Sema4D-blocking antibodies and by Sema4D knockdown. Furthermore, knocking down Sema4D by lentiviral expression of Sema4D shRNA reduces dramatically the size and vascularity of HNSCC tumor xenografts. These findings indicate that expression of Sema4D is a frequently used strategy by which a wide variety of carcinomas may promote angiogenesis, and therefore is a possible therapeutic target for the treatment of these malignancies.

Expression and function of potassium channels in the human placental vasculature

In the placental vasculature, where oxygenation may be an important regulator of vascular reactivity, there is a paucity of data on the expression of potassium (K) channels, which are important mediators of vascular smooth muscle tone. We therefore addressed the expression and function of several K channel subtypes in human placentas. The expression of voltage-gated (Kv)2.1, KV9.3, large-conductance Ca2+-activated K channel (BKCa), inward-rectified K+ channel (KIR)6.1, and two-pore domain inwardly rectifying potassium channel-related acid-sensitive K channels (TASK)1 in chorionic plate arteries, veins, and placental homogenate was assessed by RT-PCR and Western blot analysis. Functional activity of K channels was assessed pharmacologically in small chorionic plate arteries and veins by wire myography using 4-aminopyridine, iberiotoxin, pinacidil, and anandamide. Experiments were performed at 20, 7, and 2% oxygen to assess the effect of oxygenation on the efficacy of K channel modulators. KV2.1, KV9.3, BKCa, KIR6.1, and TASK1 channels were all demonstrated to be expressed at the message level. KV2.1, BKCa, KIR6.1, and TASK1 were all demonstrated at the protein level. Pharmacological manipulation of voltage-gated and ATP-sensitive channels produced the most marked modifications in vascular tone, in both arteries and veins. We conclude that K channels play an important role in controlling placental vascular function.

Elevated metabotropic glutamate receptor expression: A novel finding in invasive breast cancer

Background: Genetic mutations in key cellular pathways are necessary steps in cancer pathogenesis, and targeted manipulation of these altered pathways provides the next opportunity for therapeutic advances in controlling these malignancies. The metabotropic glutamate receptors (Grm) mediate a diverse array of cellular signaling responses including hormone, neurotransmitter, chemokine, autocrine and paracrine factors. Previous reports demonstrated chemotherapy resistance to 5-FU in colon cancer cells over-expressing Grm was reversible by administration of a Grm antagonist, and our laboratory has previously demonstrated a direct correlation between Grm over-expression and melanoma pathogenesis. This report expands these findings with novel observations in invasive human breast cancer. Methods: Human breast cancer tissue specimens (50) and matched normal breast tissue control samples Grm1 (Grm Group I: Phospholipase C stimulator) and Grm4 (Grm Group III: Adenylate cyclase inhibitor) expression. mRNA samples were reverse-transcribed to cDNA and message levels determined by quantitative real-time PCR (QRT-PCR) using a Cepheid SmartCycler and TaqMan probes. Triplicate Grm1 and Grm4 samples vs. β-actin were evaluated and ratios calculated using the ΔCt method. A 360 paired-sample invasive breast cancer tissue microarray from our practice was utilized to evaluate Grm1 and Grm4 expression using immuno-histochemical staining with anti-Grm1 and Grm4 antibodies. Results: Invasive breast cancer tissue specimens demonstrated significantly increased levels of both Grm1 (cancers: 15.7±5.5 vs normal: 2.1±0.9, p=0.011) and Grm4 (cancers: 22.3±7.5 vs normal: 1.3±0.5, p=0.005) message level when compared to matched normal breast tissue controls. Statistical comparison wascalculated using a 2-tailed Wilcoxon signed ranks test. Data are represented as mean ± std. error of the mean, and are summarized in Fig 1. Concomitant with QRT-PCR data, tissue microarray immuno-histochemistry identified Grm1 (50%) and Grm4 (60%) expression in invasive breast cancers. Conclusions: Grm over-expression has been observed in several malignancies. Our QT-PCR findings provide novel evidence of Grm over-expression in breast cancer, and may represent an important step in breast cancer pathogenesis. Immuno-histochemistry provides a readily available method for evaluating clinical specimens. These data have stimulated our investigation of Grm expression and other tumor receptor, oncogene, histologic and clinical characteristics, and utilization of available of Grm antagonists for potential targeted therapy in this tumor type.

Validating an Approach to Examining Cognitive Engagement Within Online Groups

Tools for measuring cognitive engagement within online groups have been concerned only with measuring an individual participant's cognitive engagement, without any concern for measuring cognitive engagement within groups. There remains a serious need for a scheme that measures cognitive engagement of groups and the validation of such a scheme against existing methods. The SQUAD (coding categories that are being measured, a semistructured approach for scaffolding online groups' engagement) approach to computer-mediated communication (CMC) discourse invites students within their respective groups to post messages based on five given categories: (a) suggestion, (b) question, (c) unclassified, (d) answer, and (e) delivery. In this article, the authors validated the SQUAD approach at the message level with an established framework called the practical inquiry model for assessing cognitive presence of CMC discourse. They adopted the alignments suggested by one of the developers of the Transcript Analysis Tool at sentence level to assess students' cognitive engagement within online groups in three case studies presented in this article. The authors argue that the cognitive presence attributed to the SQUAD approach has been empirically validated with respect to cognitive engagement within groups online.

Tuberin and hamartin are aberrantly expressed and linked to clinical outcome in human breast cancer: The role of promoter methylation of TSC genes

Purpose The tuberous sclerosis (TSC) genes TSC1 and TSC2 encode the protein products hamartin and tuberin, respectively, and are putative tumour suppressor genes. Germ-line mutation of either TSC gene leads to the development of the heritable disorder TSC. This disorder is characterized by the development of hamartomas in many organs and is associated with the proliferative lung disease, lymphangioleiomyomatosis, the brain tumour giant cell astrocytoma and occasionally with renal cell carcinoma. However, the TSC genes have not been studied in breast cancer. The current study investigated the expression of the TSC gene products and the potential mechanisms of their aberrancy in human breast cancer cells and tissues. Experimental design and results Using immunohistochemical analysis, both hamartin and tuberin were found to be strongly stained in normal mammary epithelial cells and weakly in stromal cells. In invasive tumour tissues, however, the staining of both proteins were to be markedly reduced ( P < 0.01). At message level, although normal and tumour tissues expressed both TSC products, the transcript levels of tuberin was significantly lower in tumour tissues compared with normal tissues ( P < 0.05). There was no statistical difference between node negative and node positive tumours with both hamartin and tuberin. Tumours from patients who developed recurrence and died from breast cancer had significantly low levels of tuberin compared with those who remained disease free ( P = 0.03 and 0.05, respectively). Likewise, hamartin levels were significantly lower in patients with metastasis, recurrence and mortality, when compared with those remained disease free ( P = 0.001, 0.041 and 0.003, respectively). Using methylation specific PCR, the TSC1 promoter was found to be heavily methylated in ZR751, MDA MB 435, and BT549, but not in MCF-7 which expressed highly level of hamartin. TSC1 promoter methylation was also seen in most breast tumours, but only in a limited number of normal tissues. The methylation of TSC2 promoter appears to be less frequent. MDA MB 468, MDA MB 483, MDA MB 435S and weakly MDA MB 435 were found to have methylated TSC2 promoter. In breast tissues, however, a very small number of samples were found to have methylation of the TSC2 promoter. Conclusion TSC1 genes are aberrantly expressed in human breast cancer cell lines and breast tumour tissues and their promoters are seen to be methylated in breast tumour tissues. The expression of TSC1 is associated with an unfavourable clinical outcome in patients with breast cancer.

AtPDR12 Contributes to Lead Resistance in Arabidopsis

Arabidopsis (Arabidopsis thaliana) contains about 130 ATP-binding cassette (ABC) proteins, which are likely to contribute to the transport of diverse materials, including toxic substances. However, the substrates of ABC transporters remain unknown in most cases. We tested which ABC transporter is involved in detoxification of lead [Pb(II)]. Among the many tested, we found that the message level of only AtPDR12 increased in both shoots and roots of Pb(II)-treated Arabidopsis, suggesting that it may be involved in the detoxification of Pb(II). AtPDR12-knockout plants (atpdr12) were used to further test this possibility. In Pb(II)-containing medium, atpdr12 plants grew less well and had higher Pb contents than those of wild-type plants. In contrast, AtPDR12-overexpressing Arabidopsis plants were more resistant to Pb(II) and had lower Pb contents than wild-type plants. The mutant phenotypes and their Pb contents, as well as the localization of the GFP:AtPDR12 fusion protein at the plasma membrane, suggest that AtPDR12 functions as a pump to exclude Pb(II) and/or Pb(II)-containing toxic compounds from the cytoplasm. Inhibition of glutathione synthesis by addition of buthionine sulfoximine to the growth medium exacerbated the Pb(II)-sensitive phenotype of atpdr12 plants, consistent with a glutathione-dependent detoxification mechanism operating in parallel with an AtPDR12-dependent mechanism. Thus, we propose that AtPDR12 is an ABC transporter that contributes to Pb(II) resistance in Arabidopsis.

What Is Targeted Therapy?

This Special Series issue of the Journal of Clinical Oncology includes two papers discussing new targeted therapies for breast cancer. Clinical and laboratory investigators interested in breast cancer will find these subjects both appropriate and timely. Targeted therapies in breast cancer (especially those targeting the estrogen receptor and HER-2) have transformed the face of this disease over the past generation. The very term ‘‘targeted therapy’’ raises questions deserving of our consideration. What do we mean by targeted therapy? Functionally, how do we ensure the success of targeted therapy in the clinic? Is the phrase anything more than another medical cliche, like the word ‘‘paradigm’’? At its simplest, targeted therapy implies a therapy with a specificmolecular target.This, itmustbesaid, is avery lowlevel definition. Any therapy that worksmust have amolecular target. In some cases (trastuzumab), we discover the target first, while in others (aspirin), we discover the drug before the target. In its day, fluorouracil was held up as targeted therapy, and appropriately so. In a related way, with targeted therapy, do wemean that the therapy has only one target? Some of our best targeted therapies (eg, imatinib) have more than one molecular target, so we cannot even claim that targeted therapy requires an exacting degree of specificity. So the simple equation, ‘‘drug molecular target Z targeted therapy,’’ is essentially meaningless. At best, it can be considered necessary but not sufficient. Breast cancer supplies a better functional definition of targeted therapy. In addition to the existence of a target (the estrogen receptor for tamoxifen, HER-2 for trastuzumab), the breast cancer story tells us what we should require of targeted therapies. A targeted therapy should attack a biologically important process (usually, though not necessarily, a single molecule), preferably one central to a hallmark of cancer. The target should bemeasurable in the clinic, and measurement of the target (in either quantitative or qualitative terms) should correlate with clinical outcome when the targeted therapy is administered. Using this approach, gefitinib moved into the realm of targeted therapy only in the clinic. That is to say, its inclusion in the targeted therapy club required the discovery of epidermal growth factor mutations correlating with therapeutic outcome in lung cancer patients. Translational science is often thought of in terms of an arrow directed from the lab to the clinic. As I have argued, this unidirectional arrow is simplistic (ie, gefitinib epidermal growth factor receptor Z targeted therapy), useless (the great majority of treated patients failed to benefit from what the laboratory called ‘‘targeted’’ therapy), and naive (a generation of cell-line work having failed to detect the existence of epidermal growth factor mutations in clinical lung cancer specimens). Targeted therapy requires clinical validation, or it is not targeted therapy. Similarly, it is reasonable to suggest that agents targeting vascular endothelial growth factor do not yet pass the bar of targeted therapy. As Schneider and Miller demonstrate, we are currently unable to measure a target (in breast cancer or any other disease) correlating treatment with outcome. We are not even certain at present whether the cells being attacked are endothelial cells, cancer cells, or both. At present, antiangiogenic therapy remains something less than targeted therapy in a functional sense. This is not to say that antiangiogenic therapy may not yet become targeted therapy: multiple surrogate markers of the angiogenic process are currently under investigation at the message, protein, and organ level (with genebased, immunohistochemical, and molecular imaging approaches). No doubt we will discover a great deal more about therapeutic targeting of antiangiogenic agents in coming years; our impatience in this regard should not blind us to the reality that the first definably antiangiogenic agent (bevacizumab) received US Food and Drug Administration approval (for colorectal cancer) in the very recent past. Indeed, if what I have said regarding a definition of targeted therapy is correct, it is premature to consider a therapy functionally targeted unless, and until, large positive studies become available. As Schneider VOLUME 23 d NUMBER 8 d MARCH 1

Natural killer cells play a role in MHC class I in vivo induction in tumor cells that are MHC negative in vitro

The natural killer (NK) cell is one of the key cells in discriminating major histocompatibility complex (MHC) negative 'missing-self' target tumor cells, and interleukin 2 (IL-2) treatment was effective in inducing NK cell activation. In this study, we tried to clarify how poorly-immunogenic murine B16 melanoma could be discriminated in vivo by creating an IL-2 cDNA-transduced immunogene therapy model (B16/IL-2). In vitro study showed that IL-2 introduction did not induce MHC class I. However, immune cells depleted total tumor digest, which consisted of 90% anti-melanoma MM2-9B6-positive cells that revealed B16/IL-2 strongly, and control tumor cells (B16/mock) partially expressed MHC class I in vivo. In the B16/IL-2 model, NK cell infiltration was 10 times higher than B16/mock (7.6 versus 0.73, p=0.017). In addition, the cell surface of CD69-expressing NK cell population was increased in B16/IL-2, and the interferon gamma (IFNgamma) message level in NK cells was significantly increased in B16/IL-2 (p=0.0359). Interestingly, NK cell depletion in vivo completely abolished MHC class I expression on B16/mock, and decreased MHC class I expression and T-cell infiltration in B16/IL-2. These data suggest that NK cells are not only important for missing-self recognition, but are also crucial for induction of tumor cell MHC molecule expression and play an important role in helping acquired immunity to recognize tumor cells.

Ethylene‐binding activity, gene expression levels, and receptor system output for ethylene receptor family members from Arabidopsis and tomato†

Ethylene signaling in plants is mediated by a family of ethylene receptors related to bacterial two-component regulators. Expression in yeast of ethylene-binding domains from the five receptor isoforms from Arabidopsis thaliana and five-receptor isoforms from tomato confirmed that all members of the family are capable of high-affinity ethylene-binding activity. All receptor isoforms displayed a similar level of ethylene binding on a per unit protein basis, while members of both subfamily I and subfamily II from Arabidopsis showed similar slow-release kinetics for ethylene. Quantification of receptor-isoform mRNA levels in receptor-deficient Arabidopsis lines indicated a direct correlation between total message level and total ethylene-binding activity in planta. Increased expression of remaining receptor isoforms in receptor-deficient lines tended to compensate for missing receptors at the level of mRNA expression and ethylene-binding activity, but not at the level of receptor signaling, consistent with specialized roles for family members in receptor signal output.

Effect of Long-Term Inhalation of Toner on Extracellular Matrix in the Lungs of Rats In Vivo

We assessed the effects of long-term inhalation of toner on the pathological changes and gene expression with the synthesis and degradation of collagenous extracellular matrix in a rat model. Female Wistar rats (10 wk old) were divided evenly into a high concentration exposure group (H: 15.2 mg/m3), a low concentration exposure group (L: 5.5 mg/m3), and a control group. The mass median aerodynamic diameter of toner was 4.5 μm. The rats were sacrificed at the termination of a 1-yr or 2-yr inhalation period. Pathological examination was performed from the left lung, and transcriptional levels of mRNA extracted from the right lung were assessed by semiquantitative reverse-transcription polymerase chain polymerase (RT-PCR). The pathological findings showed mild pulmonary fibrosis in 20% (L, 1 yr), 40% (H, 1 yr), 56% (L, 2 yr) and 62% (H, 2 yr), while lung cancer was not observed in any of the exposed groups. In the 1-yr high-concentration group, gene expression of matrix metalloproteinase-2 (MMP-2) and type I collagen mRNA in the rat lungs increased, while tissue inhibitors of metalloproteinase-2 (TIMP-2) decreased. The 2-yr high-concentration group increased in message level of type I collagen and TIMP-2 but not that of MMP-2. These data suggested that results of gene expression of MMP, TIMP, and collagen in the 2-yr exposure may lead to accumulation of collagen compared to the 1-yr exposure, and that the imbalance of the expression of MMPs, TIMPs, and extracellular matrix might be associated with pulmonary fibrosis induced by toner.