Search

Background Cardio-facio-cutaneous (CFC) syndrome is a human multiple congenital anomaly syndrome that is caused by activating heterozygous mutations in either BRAF, MEK1, or MEK2, three protein kinases of the Ras/mitogen-activated protein kinase (MAPK) pathway. CFC belongs to a group of syndromes known as RASopathies. Skeletal muscle hypotonia is a ubiquitous phenotype of RASopathies, especially in CFC syndrome. To better understand the underlying mechanisms for the skeletal myopathy in CFC, a mouse model with an activating BrafL597V allele was utilized. Results The activating BrafL597V allele resulted in phenotypic alterations in skeletal muscle characterized by a reduction in fiber size which leads to a reduction in muscle size which are functionally weaker. MAPK pathway activation caused inhibition of myofiber differentiation during embryonic myogenesis and global transcriptional dysregulation of developmental pathways. Inhibition in differentiation can be rescued by MEK inhibition. Conclusions A skeletal myopathy was identified in the CFC BrafL597V mouse validating the use of models to study the effect of Ras/MAPK dysregulation on skeletal myogenesis. RASopathies present a novel opportunity to identify new paradigms of myogenesis and further our understanding of Ras in development. Rescue of the phenotype by inhibitors may help advance the development of therapeutic options for RASopathy patients.

Costello syndrome (CS) is a congenital disorder caused by heterozygous activating germline HRAS mutations in the canonical Ras/mitogen-activated protein kinase (Ras/MAPK) pathway. CS is one of the RASopathies, a large group of syndromes caused by mutations within various components of the Ras/MAPK pathway. An important part of the phenotype that greatly impacts quality of life is hypotonia. To gain a better understanding of the mechanisms underlying hypotonia in CS, a mouse model with an activating HrasG12V allele was utilized. We identified a skeletal myopathy that was due, in part, to inhibition of embryonic myogenesis and myofiber formation, resulting in a reduction in myofiber size and number that led to reduced muscle mass and strength. In addition to hyperactivation of the Ras/MAPK and PI3K/AKT pathways, there was a significant reduction in p38 signaling, as well as global transcriptional alterations consistent with the myopathic phenotype. Inhibition of Ras/MAPK pathway signaling using a MEK inhibitor rescued the HrasG12V myopathy phenotype both in vitro and in vivo, demonstrating that increased MAPK signaling is the main cause of the muscle phenotype in CS., Summary: A Costello syndrome (CS) mouse model carrying a heterozygous Hras p.G12V mutation was utilized to investigate Ras pathway dysregulation, revealing that increased MAPK signaling is the main cause of the muscle phenotype in CS.

There is a need to develop novel targeted imaging and therapeutic agents that can aid in early diagnosis, detection of metastasis and treatment of melanoma. Alpha-3 integrin is overexpressed in 82% of metastatic melanomas in humans and may be a potential target for peptide ligands carrying therapeutic agents. Five melanoma cell lines were generated from canine primary oral and metastatic canine tumors, grown in mice, and validated with melanoma markers Melan A, S-100, Micropthalmia transcription factor (MITF), Tyrosinase, and MART-1. The melanoma cell lines were tested for binding affinity to previously published alpha-3 integrin-binding peptides containing the cdGXGXXc motif. Fluorescent conjugates of the alpha-3 integrin binding OA02 peptide were used to quantify receptor affinity in the cell lines, a specimen of canine primary oral melanoma, and melanoma xenografts. Alpha-3 integrin was expressed by all 5 canine melanoma cell lines. Four of the 5 lines as well as the primary canine tumor showed affinity to alpha-3 integrin binding peptides with the cdGXGXXc motif. Optical imaging of canine melanoma xenografts in nude mice indicates rapid, strong uptake of the optical tracer in the tumor with an average persistence of approximately 48 hours. Ex vivo images showed high tumor-to-background ratio, with tumor signals more than twice that of the kidney and other vital organs. We propose that integrin alpha-3 integrin binding ligands could potentially become useful probes for imaging and delivery of cytotoxic agents for the treatment of melanoma.

Accumulations of hypertrophic, intensely glial fibrillary acidic protein-positive (GFAP+) astroglia, which also express immunoreactive nestin and vimentin, are prominent features of multiple sclerosis lesions. The issues of the cellular origin of hypertrophic GFAP+/vimentin+/nestin+“reactive” astroglia and also the plasticities and lineage relationships among three macroglial progenitor populations—oligodendrocyte progenitor cells (OPCs), astrocytes and ependymal cells—during multiple sclerosis and other CNS diseases remain controversial. We used genetic fate-mappings with a battery of inducible Cre drivers (Olig2-Cre-ERT2, GFAP-Cre-ERT2, FoxJ1-Cre-ERT2and Nestin-Cre-ERT2) to explore these issues in adult mice with myelin oligodendrocyte glycoprotein peptide-induced experimental autoimmune encephalomyelitis (EAE). The proliferative rate of spinal cord OPCs rose fivefold above control levels during EAE, and numbers of oligodendroglia increased as well, but astrogenesis from OPCs was rare. Spinal cord ependymal cells, previously reported to be multipotent, did not augment their low proliferative rate, nor give rise to astroglia or OPCs. Instead, the hypertrophic, vimentin+/nestin+, reactive astroglia that accumulated in spinal cord in this multiple sclerosis model were derived by proliferation and phenotypic transformation of fibrous astroglia in white matter, and solely by phenotypic transformation of protoplasmic astroglia in gray matter. This comprehensive analysis of macroglial plasticity in EAE helps to clarify the origins of astrogliosis in CNS inflammatory demyelinative disorders.

The αvβ3 integrin, expressed on the surface of various normal and cancer cells, is involved in numerous physiologic processes such as angiogenesis, apoptosis, and bone resorption. Because this integrin plays a key role in angiogenesis and metastasis of human tumors, αvβ3 integrin ligands are of great interest to advances in targeted therapy and cancer imaging. In this report, one-bead one-compound (OBOC) combinatorial libraries containing the arginine-glycine-aspartic acid (RGD) motif were designed and screened against K562 myeloid leukemia cells that had been transfected with the human αvβ3 integrin gene. Cyclic peptide LXW7 was identified as a leading ligand with a built-in handle that binds specifically to αvβ3 and showed comparable binding affinity (IC50 = 0.68 ± 0.08 μmol/L) to some of the well-known RGD “head-to-tail” cyclic pentapeptide ligands reported in the literature. The biotinylated form of LXW7 ligand showed similar binding strength as LXW7 against αvβ3 integrin, whereas biotinylated RGD cyclopentapeptide ligands revealed a 2- to 8-fold weaker binding affinity than their free forms. LXW7 was able to bind to both U-87MG glioblastoma and A375M melanoma cell lines, both of which express high levels of αvβ3 integrin. In vivo and ex vivo optical imaging studies with the biotinylated ligand/streptavidin-Cy5.5 complex in nude mice bearing U-87MG or A375M xenografts revealed preferential uptake of biotinylated LXW7 in tumor. When compared with biotinylated RGD cyclopentapeptide ligands, biotinylated LXW7 showed higher tumor uptake but lower liver uptake. Mol Cancer Ther; 9(10); 2714–23. ©2010 AACR.

Previous studies have shown that oligodendroglial progenitor cells (OPCs) can give rise to neuronsin vitroand in perinatal cerebral cortexin vivo. We now report that OPCs in adult murine piriform cortex express low levels of doublecortin, a marker for migratory and immature neurons. Additionally, these OPCs express Sox2, a neural stem cell marker, and Pax6, a transcription factor characteristic of progenitors for cortical glutamatergic neurons. Genetic fate-mapping by means of an inducible Cre–LoxP recombination system proved that these OPCs differentiate into pyramidal glutamatergic neurons in piriform cortex. Several lines of evidence indicated that these newly formed neurons became functionally integrated into the cortical neuronal network. Our data suggest that NG2+/PDGFRα+proteolipid protein promoter-expressing progenitors generate pyramidal glutamatergic neurons within normal adult piriform cortex.

Genetic or pharmacological activation of canonical Wnt/β-catenin signaling inhibits oligodendrocyte differentiation. Transcription factor 7-like 2 (TCF7l2), also known as TCF4, is a Wnt effector induced transiently in the oligodendroglial lineage. A well accepted dogma is that TCF7l2 inhibits oligodendrocyte differentiation through activation of Wnt/β-catenin signaling. We report that TCF7l2 is upregulated transiently in postmitotic, newly differentiated oligodendrocytes. Usingin vivogene conditional ablation, we found surprisingly that TCF7l2 positively regulates neonatal and postnatal mouse oligodendrocyte differentiation during developmental myelination and remyelination in a manner independent of the Wnt/β-catenin signaling pathway. We also reveal a novel role of TCF7l2 in repressing a bone morphogenetic protein signaling pathway that is known to inhibit oligodendrocyte differentiation. Thus, our study provides novel data justifying therapeutic attempts to enhance, rather than inhibit, TCF7l2 signaling to overcome arrested oligodendroglial differentiation in multiple sclerosis and other demyelinating diseases.

PKD2, the second gene for the autosomal dominant polycystic kidney disease (ADPKD), encodes a protein, polycystin-2, with predicted structural similarity to cation channel subunits. However, the function of polycystin-2 remains unknown. We used polyclonal antisera specific for the intracellular NH(2) and COOH termini to identify polycystin-2 as an approximately 110-kDa integral membrane glycoprotein. Polycystin-2 from both native tissues and cells in culture is sensitive to Endo H suggesting the continued presence of high-mannose oligosaccharides typical of pre-middle Golgi proteins. Immunofluorescent cell staining of polycystin-2 shows a pattern consistent with localization in the endoplasmic reticulum. This finding is confirmed by co-localization with protein-disulfide isomerase as determined by double indirect immunofluorescence and co-distribution with calnexin in subcellular fractionation studies. Polycystin-2 translation products truncated at or after Gly(821) retain their exclusive endoplasmic reticulum localization while products truncated at or before Glu(787) additionally traffic to the plasma membrane. Truncation mutants that traffic to the plasma membrane acquire Endo H resistance and can be biotinylated on the cell surface in intact cells. The 34-amino acid region Glu(787)-Ser(820), containing two putative phosphorylation sites, is responsible for the exclusive endoplasmic reticulum localization of polycystin-2 and is the site of specific interaction with an as yet unidentified protein binding partner for polycystin-2. The localization of full-length polycystin-2 to intracellular membranes raises the possibility that the PKD2 gene product is a subunit of intracellular channel complexes.

The expression of the gut tumor suppressor gene adenomatous polyposis coli (Apc) and its role in the oligodendroglial lineage are poorly understood. We found that immunoreactive APC is transiently induced in the oligodendroglial lineage during both normal myelination and remyelination following toxin-induced, genetic, or autoimmune demyelination murine models. Using the Cre/loxP system to conditionally ablate APC from the oligodendroglial lineage, we determined that APC enhances proliferation of oligodendroglial progenitor cells (OPCs) and is essential for oligodendrocyte differentiation in a cell-autonomous manner. BiallelicApcdisruption caused translocation of β-catenin into the nucleus and upregulated β-catenin-mediated Wnt signaling in early postnatal but not adult oligodendroglial lineage cells. The results of conditional ablation ofApcorCtnnb1(the gene encoding β-catenin) and of simultaneous conditional ablation ofApcandCtnnb1revealed that β-catenin is dispensable for postnatal oligodendroglial differentiation, thatApcone-allele deficiency is not sufficient to dysregulate β-catenin-mediated Wnt signaling in oligodendroglial lineage cells, and that APC regulates oligodendrocyte differentiation through β-catenin-independent, as well as β-catenin-dependent, mechanisms. Gene ontology analysis of microarray data suggested that the β-catenin-independent mechanism involves APC regulation of the cytoskeleton, a result compatible with established APC functions in neural precursors and with our observation thatApc-deleted OPCs develop fewer, shorter processesin vivo. Together, our data support the hypothesis that APC regulates oligodendrocyte differentiation through both β-catenin-dependent and additional β-catenin-independent mechanisms.

Pharmacological studies have suggested that oligodendroglial N-methyl-D-aspartate glutamate receptors (NMDARs) mediate white matter injury in a variety of CNS diseases, including multiple sclerosis (MS). We tested this hypothesis in experimental autoimmune encephalomyelitis (EAE), a model of human MS, by timed conditional disruption of oligodendroglial NR1, an essential subunit of functional NMDARs, using an inducible proteolipid protein (Plp) promoter-driven Cre-loxP recombination system. We found that selective ablation of oligodendroglial NR1 did not alter the clinical severity of EAE elicited in C57BL/6 mice by immunization with myelin oligodendrocyte glycoprotein peptide 35–55 (MOG-peptide), nor were there significant differences between the oligodendroglial NR1 KO and non-KO mice in numbers of axons lost in spinal cord dorsal funiculi or severity of spinal cord demyelination. Similarly, constitutive deletion of NR3A, a modulatory subunit of oligodendroglial NMDARs, did not alter the course of MOG-peptide EAE. Furthermore, conditional and constitutive ablation of NR1 in neonatal oligodendrocyte progenitor cells (OPCs) did not interrupt their normal maturation and differentiation. Collectively, our data suggest that oligodendroglial lineage NMDARs are neither required for timely postnatal development of the oligodendroglial lineage, nor significant participants in the pathophysiology of MOG-peptide EAE.

Two new migrated hopane triterpenoids, viz. 4alpha-hydroxyfilican-3-one and fern-9(11)-en-12beta-ol, and olean-18-en-3-one and olean-12-en-3-one as the first example of oleanane compounds from Adiantum ferns were isolated along with many other known triterpenoids from Adiantum capillus-veneris of China and Egypt. Their structures were elucidated by spectroscopic analyses.

Background/Aims Early detection and identification of bacteria in ascitic fluid could result in more timely treatment of cirrhotic patients with spontaneous bacterial peritonitis (SBP) or subclinical SBP. The aim of this study was to evaluate the usefulness of a bacterial DNA microarray for the rapid diagnosis of SBP and rapid bacterial identification in cirrhotic patients with ascites. Methods Thirty-seven cirrhotic patients with ascites (25 men and 12 women) participated. Ascitic fluid obtained from patients was tested by the bacterial DNA microarray method and by the conventional culture method. Results SBP and bacterascites were diagnosed in 8 (16.7%) of 48 specimens by the conventional method. The bacterial DNA microarray proved the existence of bacteria in 6 (75%) of 8 samples with SBP or bacterascites using the conventional method as a gold standard. A corresponding rate of bacterial species identification between the two methods was found in 5 of 6 samples (83.3%). It took 1.47±0.96 and 5.14±2.6 days to receive the data by the microarray and conventional method, respectively (p

Polycystin-2 (PC2), the gene product of one of two genes mutated in dominant polycystic kidney disease, is a member of the transient receptor potential cation channel family and can function as intracellular calcium (Ca 2+ ) release channel. We performed a yeast two-hybrid screen by using the NH 2 terminus of PC2 and identified syntaxin-5 (Stx5) as a putative interacting partner. Coimmunoprecipitation studies in cell lines and kidney tissues confirmed interaction of PC2 with Stx5 in vivo. In vitro binding assays showed that the interaction between Stx5 and PC2 is direct and defined the respective interaction domains as the t-SNARE region of Stx5 and amino acids 5 to 72 of PC2. Single channel studies showed that interaction with Stx5 specifically reduces PC2 channel activity. Epithelial cells overexpressing mutant PC2 that does not bind Stx5 had increased baseline cytosolic Ca 2+ levels, decreased endoplasmic reticulum (ER) Ca 2+ stores, and reduced Ca 2+ release from ER stores in response to vasopressin stimulation. Cells lacking PC2 altogether had reduced cytosolic Ca 2+ levels. Our data suggest that PC2 in the ER plays a role in cellular Ca 2+ homeostasis and that Stx5 functions to inactivate PC2 and prevent leaking of Ca 2+ from ER stores. Modulation of the PC2/Stx5 interaction may be a useful target for impacting dysregulated intracellular Ca 2+ signaling associated with polycystic kidney disease.

Purpose: We previously reported the usefulness of a qualified highly sensitive detection method for human telomerase reverse transcriptase (hTERT) mRNA in serum with 89.7% sensitivity for hepatocellular carcinoma (HCC). In this study, we developed a quantitative detection method for serum hTERT mRNA and examined the clinical significance in HCC diagnosis. Experimental Background: In 64 patients with HCC, 20 with liver cirrhosis, 20 with chronic hepatitis, and 50 healthy individuals, we measured serum hTERT mRNA by using the newly developed real-time quantitative reverse transcription-PCR with SYBR Green I. We examined its sensitivity and specificity in HCC diagnosis, clinical significance in comparison with other tumor markers, and its correlations with the clinical variables by using multivariate analyses. Results: Serum hTERT mRNA showed higher values in patients with HCC than those with chronic liver diseases. hTERT mRNA expression was shown to be independently correlated with clinical variables such as tumor size, number, and degree of differentiation (P < 0.001, each). The sensitivity/specificity of hTERT mRNA and alpha;-fetoprotein (AFP) mRNA in HCC diagnosis were 88.2%/70.0% for hTERT and 71.6%/67.5% for AFP, respectively. hTERT mRNA proved to be superior to AFP mRNA, AFP, and des-γ-carboxy prothrombin in HCC diagnosis. Furthermore, hTERT mRNA in serum was associated with that in HCC tissue. Conclusions: The usefulness of hTERT mRNA expression in HCC diagnosis and its superiority to conventional tumor markers were shown. Therefore, serum hTERT mRNA is a novel and available marker for HCC diagnosis.

We report a case involving well-differentiated hepatocellular carcinoma (HCC) developing to HCC with sarcomatous changes after radiofrequency ablation (RFA). In a cirrhotic patient with both hepatitis B surface antigen and hepatitis C virus RNA, a well-differentiated HCC with a diameter of 2 cm was detected in segment IV of the liver. A combination of transcatheter arterial embolization and percutaneous ethanol injection (PEI) was performed and, after 8 months, PEI was performed for recurrent tumors. Fifteen months after the first treatment, a recurrent tumor measuring 3.5 cm in diameter was detected in segment IV, which was demonstrated as well-differentiated HCC by tumor biopsy, and treated by RFA. Although the treated lesion was reduced to 2.5 cm in diameter 6 months after RFA, the tumor rapidly enlarged to 6 cm in diameter 2 months later and progressed to lymph node metastasis. Aspiration biopsy showed spindle-shaped sarcomatoid cells with positive staining for both vimentin and keratin. The patient died of HCC progression 10 months after RFA. Autopsy findings showed both sarcomatoid cells and trabecular HCC cells. The sarcomatoid cells had metastasized to the lymph nodes and distant organs. This is the first case illustrating a sarcomatous HCC after RFA. Of interest, RFA may be related to the development of sarcomatous HCC.

Objectives: We investigated how radiofrequency ablation (RFA) alone or the combination with transcatheter arterial embolization (TAE-RFA) for hepatocellular carcinoma (HCC) affects the liver parenchymal function of underlying chronic liver disease to find a predictive factor for selecting appropriate candidates for RFA. Methods: In 53 HCC patients (RFA alone, 25; TAE-RFA, 28), liver laboratory tests and development of complications were monitored until 6 months after treatments. Serum albumin deteriorated within 1 month and recovered after 6 months in both groups, whereas the elevation of the Pugh score continued in RFA alone group and that of serum bilirubin continued in TAE-RFA group until 6 months. The elevation of Pugh score 6 months after RFA was significantly more frequent in patients with a high pre-treatment Pugh score (/=8 points) than in those with a low pre-treatment Pugh score (8 points). Complications after RFA were observed in 11 of the 53 patients: temporary complications in 5, refractory ascites in 6. Refractory ascites developed in only patients with a high pre-treatment Pugh score. Conclusion: Liver parenchymal functions in patients with a low pre-treatment Pugh score were transiently deteriorated by RFA or TAE-RFA. However, in patients with a high Pugh score, RFA or TAE-RFA induces long-term deterioration of the liver parenchymal functions and causes serious complications. Therefore, patients with a Pugh score/=8 points would not be good candidates for RFA or TAE-RFA.

Polycystin-2, the product of the gene mutated in type 2 autosomal dominant polycystic kidney disease (ADPKD), is the prototypical member of a subfamily of the transient receptor potential (TRP) channel superfamily, which is expressed abundantly in the endoplasmic reticulum (ER) membrane. Here, we show by single channel studies that polycystin-2 behaves as a calcium-activated, high conductance ER channel that is permeable to divalent cations. Epithelial cells overexpressing polycystin-2 show markedly augmented intracellular calcium release signals that are lost after carboxy-terminal truncation or by the introduction of a disease-causing missense mutation. These data suggest that polycystin-2 functions as a calcium-activated intracellular calcium release channel in vivo and that polycystic kidney disease results from the loss of a regulated intracellular calcium release signalling mechanism.

Background The gender-specific influences of various confounding factors, including apnea, age, BMI, and cigarette consumption, on the function of the parasympathetic nerve system (PNS) during sleep in OSA patients has never been investigated. Methods One hundred ninety-seven males and 63 females with OSA were subjected to full PSG examinations including assessment of R-R intervals (RRIs) during an overnight ECG. The PNS-derived modulatory effect on the RRIs and the variability of this effect were quantified during REM and NREM using instantaneous time-frequency analysis with complex demodulation. The spectral domain with the maximum instantaneous amplitude in the high-frequency band between 0.15 and 0.4 Hz was defined as the main HF peak and used as a surrogate marker of PNS discharge. Based on density-spectrum-array maps of the main HF peaks (HF-DSA map), shifts in the central frequency of the main HF peak over time were continuously observed. When the main HF peaks on the HF-DSA maps maintained the same central frequency for more than 20 sec or 5 min, the PNS functions were considered to be “stable” or “very stable”, respectively. Results Apneas enhanced PNS-derived cardiac-modulation during REM in males, but more importantly, they made PNS-function unstable during both REM and NREM in males and during NREM in females. Aging blunted the PNS-derived cardiac-modulation during both REM and NREM regardless of gender, but aging had no impact on the stability of PNS-function. BMI blunted PNS-eliciting cardiac-modulation during REM in males and during NREM in both males and females. BMI made the PNS unstable during REM in females. Neither height nor cigarette consumption influenced any PNS-related parameter. Conclusions The PNS-derived cardiac-modulation was generally inhibited by aging and obesity, in which the effect of obesity was gender-specific. The PNS instability at nighttime was mainly induced by apneas but by obesity particularly during REM in females.

Pkd2, the mouse homologue of PKD2, the gene responsible for the second form of autosomal dominant polycystic kidney disease, is highly expressed in fetal and adult mouse tissues. The expression of Pkd2 is developmentally regulated. To begin to dissect out the regulatory mechanism of Pkd2 expression, we characterized the basic features of the gene structure and identified potential cis-regulatory elements of Pkd2 transcription. Pkd2 spans 42 kb with a transcription start site 165 bp upstream of the translation start codon. Exon 1 of Pkd2 is 755 bp long, and the full-length transcript is 5215 bp. The Pkd2 promoter region is GC-rich and lacks a consensus TATA or CCAAT box. Consensus binding sites for the transcription factors Sp-1, NF-1, and Ap-2 lie in the 5' upstream region of Pkd2. The Sp-1 binding site is conserved in 5' upstream sequences of both the mouse and the human genes. The CAT activity of a series of upstream segments from +178 to -2749 was assessed in MDCK, LLCPK1, COS-7, and HEK293 cells. Deletion analysis identified a 409-bp fragment from position -221 to +178 responsible for basal promoter activity. A 922-bp fragment from -744 to +178 showed the highest level of CAT activity in the cell lines tested. These data define a functional promoter candidate region for Pkd2.

It is estimated that approximately 15% of families with autosomal dominant polycystic kidney disease (ADPKD) have mutations in PKD2. Identification of these mutations is central to identifying functionally important regions of gene and to understanding the mechanisms underlying the pathogenesis of the disorder. The current study describes mutations in six type 2 ADPKD families. Two single base substitution mutations discovered in the ORF in exon 14 constitute the most COOH-terminal pathogenic variants described to date. One of these mutations is a nonsense change and the other encodes an apparent missense variant. Reverse transcription-PCR from patient lymphoblast RNA showed that, in addition, both mutations resulted in out-of-frame splice variants by activating cryptic splice sites via different mechanisms. The apparent missense variant produced such a strong splicing signal that the processed transcript from the mutant chromosome did not contain any of the normally spliced, missense product. A third mutation, a nonconservative missense change effecting a negatively charged residue in the third transmembrane span, is likely pathogenic and defines a highly conserved residue consistent with a potential channel subunit function for polycystin-2. The remaining three mutations included two frame shifts resulting from deletion of one or two bases in exons 6 and 10, respectively, and a nonsense mutation due to a single base substitution in exon 4. The study also defined a novel intragenic polymorphism in exon 1 that will be useful in analyzing “second hits” in PKD2. Finally, the study demonstrates that there are reduced levels of normal polycystin-2 protein in lymphoblast lines from PKD2-affected individuals and that truncated mutant polycystin-2 cannot be detected in patient lymphoblasts, suggesting that the latter may be unstable in at least some tissues. The mutations described will serve as critical reagents for future functional studies in PKD2.

Mutations in PKD2 cause autosomal dominant kidney disease (ADPKD). Polycystin-2, the PKD2 gene product, is an integral membrane glycoprotein of unknown function. We have identified PKD2L, another member of the PKD2 gene family. PKD2L is expressed in adult heart and skeletal muscle, brain, spleen, testis, and retina, and alternative transcripts of 2.4, 2.7, and 3.0 kb are seen. PKD2L shows 56% identity and 76% similarity with polycystin-2 over a 581-amino-acid span; however, the COOH-terminal 65 residues of PKD2L are unrelated to PKD2. PKD2L is localized to chromosome 10q25 and is excluded as a candidate gene for autosomal recessive polycystic kidney disease, autosomal dominant polycystic liver disease, and the third form of ADPKD. Given the high degree of homology between PKD2L and PKD2, it is likely that the respective functions of these proteins are also closely related.

Germline mutations in PKD2 cause autosomal dominant polycystic kidney disease. We have introduced a mutant exon 1 in tandem with the wild-type exon 1 at the mouse Pkd2 locus. This is an unstable allele that undergoes somatic inactivation by intragenic homologous recombination to produce a true null allele. Mice heterozygous and homozygous for this mutation, as well as Pkd+/− mice, develop polycystic kidney and liver lesions that are indistinguishable from the human phenotype. In all cases, renal cysts arise from renal tubular cells that lose the capacity to produce Pkd2 protein. Somatic loss of Pkd2 expression is both necessary and sufficient for renal cyst formation in ADPKD, suggesting that PKD2 occurs by a cellular recessive mechanism.

We are interested in the physiological roles of the sialoglycoconjugates associated with rod photoreceptor cells, which specifically metabolize sialic acids by balancing sialyltransferase with sialidase (1, 2). Defects of O- and N-linked sialoglycoconjugates may cause retinal dysplasia and retinal degeneration, respectively, in humans (3). Retinal degeneration can be experimentally induced in rats by modification of sialic acids of retinal sialoglycoconjugates (4, 5), but the pathomechanism underlying this degenerative process remains to be clarified.

Host and tumor surface glycans are important in regulating pivotal pathophysiological events during the development of cancer and tumor progression. Changes in glycosylation allow tumorigenic cells to seize control over developmental processes which allow cells to proliferate, invade, promote angiogenesis, and metastasize. Malignant transformation is accompanied by the expression of oncofetal antigens which are expressed on embryonic cells, tumor cells, and a few adult cells. Glycans common to human embryonic stem cells and cancer cells can be targeted using combinatorial chemistry. Several peptidomimetic ligands have been discovered using One Bead One Compound (OBOC) and One Bead Two Compound (OB2C) methods. These cell adhering ligands possess the potential to target tumorigenic cells and mediate intracellular signaling events by binding to high mannose glycans expressed on the surface of Jurkat, TK6, Her2 expressing MCF7, chemoresistant MCF7, HeLa, and SiHa cell lines. Three lead linear peptides with alternating natural and unnatural amino acids have been investigated for their in vivo targeting potential in the lymphoid leukemia, breast, and cervical cancer mouse xenograph models. In vitro and in vivo stem and cancer cell experiments, which demonstrate genetically altered glycan function, are currently being investigated for their anticancer potential. These novel agents may be used alone or in combination with chemoradiation and anti-angiogenesis strategies for treating cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2316. doi:10.1158/1538-7445.AM2011-2316

In the “one bead one compound” combinatorial library method, millions of In the “one bead one compound” combinatorial library method, millions of random peptides can be generated rapidly, such that each bead displays only one peptide entity. When live cancer cells are co-cultured with these peptide-bead libraries, beads with cell adhesion peptides will be covered entirely by the cells. Low passage human pancreatic cancer (PDAC cells;XPA1, XPA3) and immortalized normal pancreas cells were used for this study. Positive peptide-beads were then isolated for sequence analysis. Using this high throughput screening approach, we evaluated four different peptide libraries and identified several peptides that bind to pancreatic cancer cells. To test their binding specificity, these peptides were labeled with fluorophor or biotinylated, and used as reagents for in vivo imaging. Athymic nude mice were used to induce pancreatic xenografts and very low uptake in liver. One of the cyclic peptide (PCa-pep6) targets pancreatic xenografts selectively. Based on the antibody blocking assay, PCa-pep6 binds to alpha-2 and alpha-6 integrins which are over expressed in pancreatic cancer. Therefore it may potentially be used as lead compounds for the development of high affinity and high specificity therapeutic and imaging agents for pancreatic cancers. This work is supported by grant RO3-CA129786 Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4327.

The absence of curative therapies for recurrent forms of metastatic cancer has prompted a vigorous search for novel treatment strategies. Cancer immunotherapy uses the host immune system, either directly or indirectly, to combat cancer. The more successful cancer immunotherapies are antibody-based. In this study we hypothesized that a chemical antibody (chembody) with an intact Fc region can be constructed by conjugating tumor cell specific peptidomimetic agents (ligands) to the Fab region of human IgG (hIgG). The ligand of interest, RGD, binds effectively to αVβ3 integrin expressed on tumor cells. The resultant chembody will have the ability to opsonize the tumor cells and elicit host immune response against cancer cells by recruiting the immune effector cells. In this study, we (1) conjugate naïve hIgG with the ligand, (2) verify ligand conjugation, and (3) test the efficacy of the chembody. The 2-iminothiolane conjugation method was employed to conjugate the ligand to naïve hIgG. The conjugation was verified by performing Western blot analysis, MALDI-MS assay, and cell binding assay. The Western blot and Coomassie blue staining showed that conjugation was successful while the MALDI-MS assay showed a 1:3 conjugation ratio. Colorimetric cytotoxicity assay will be used to test the efficacy of the chembody. We expect chembodies with intact Fc region to be tumor specific, and actively recruit immune cells to the tumor site and have low immunogenicity. This novel individualized medicine approach in cancer immunotherapy can be very effective in eliminating the remaining cells responsible for relapse after chemotherapy or radiotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5634.