Altering α-dystroglycan receptor affinity of LCMV pseudotyped lentivirus yields unique cell and tissue tropism

Background

The envelope glycoprotein of lymphocytic choriomeningitis virus (LCMV) can efficiently pseudotype lentiviral vectors. Some strains of LCMV exploit high affinity interactions with α-dystroglycan (α-DG) to bind to cell surfaces and subsequently fuse in low pH endosomes. LCMV strains with low α-DG affinity utilize an unknown receptor and display unique tissue tropisms. We pseudotyped non-primate feline immunodeficiency virus (FIV) vectors using LCMV derived glycoproteins with high or low affinity to α-DG and evaluated their properties in vitro and in vivo.

Methods

We pseudotyped FIV with the LCMV WE54 strain envelope glycoprotein and also engineered a point mutation in the WE54 envelope glycoprotein (L260F) to diminish α-DG affinity and direct binding to alternate receptors. We hypothesized that this change would alter in vivo tissue tropism and enhance gene transfer to neonatal animals.

Results

In mice, hepatic α- and β-DG expression was greatest at the late gestational and neonatal time points. When displayed on the surface of the FIV lentivirus the WE54 L260F mutant glycoprotein bound weakly to immobilized α-DG. Additionally, LCMV WE54 pseudotyped FIV vector transduction was neutralized by pre-incubation with soluble α-DG, while the mutant glycoprotein pseudotyped vector was not. In vivo gene transfer in adult mice with either envelope yielded low transduction efficiencies in hepatocytes following intravenous delivery. In marked contrast, neonatal gene transfer with the LCMV envelopes, and notably with the FIV-L260F vector, conferred abundant liver and lower level cardiomyocyte transduction as detected by luciferase assays, bioluminescent imaging, and β-galactosidase staining.

Conclusions

These results suggest that a developmentally regulated receptor for LCMV is expressed abundantly in neonatal mice. LCMV pseudotyped vectors may have applications for neonatal gene transfer.

Abbreviations

Armstrong 53b (Arm53b); baculovirus Autographa californica GP64 (GP64); charge-coupled device (CCD); dystroglycan (DG); feline immunodeficiency virus (FIV); glycoprotein precursor (GP-C); firefly luciferase (Luc); lymphocytic choriomeningitis virus (LCMV); nuclear targeted β-galactosidase (ntLacZ); optical density (OD); PBS/0.1% (w/v) Tween-20 (PBST); relative light units (RLU); Rous sarcoma virus (RSV); transducing units per milliliter (TU/ml); vesicular stomatitis virus (VSV-G); wheat germ agglutinin (WGA); 50% reduction in binding (C₅₀).     

Development and characterization of highly polymorphic long TC repeat microsatellite markers for genetic analysis of peanut

Background

The immune system has paradoxical roles during cancer development and the prognostic significance of immune modulating factors is controversial. The aim of this study was to determine the expression of cyclooxygenase 2 (COX-2), transforming growth factor-beta (TGF- beta), interleukin-10 (IL-10) and their prognostic significance in breast cancers. Ki67 was included as a measure of growth fraction of tumor cells.

Methods

On immunohistochemical stained slides from 38 breast cancer patients, we performed digital video analysis of tumor cell areas and adjacent tumor stromal areas from the primary tumors and their corresponding lymph node metastases. COX-2 was recorded as graded staining intensity.

Results

The expression of TGF-beta, IL-10 and Ki67 were recorded in tumor cell areas and adjacent tumor stromal areas. In both primary tumors and metastases, the expression of COX-2 was higher in the tumor stromal areas than in the tumor cell areas (both P < 0.001). High stromal staining intensity in the primary tumors was associated with a 3.9 (95% CI 1.1-14.2) times higher risk of death compared to the low staining group (P = 0.036). The expression of TGF-beta was highest in the tumor cell areas of both primary tumors and metastases (both P < 0.001). High stromal expression of TGF-beta was associated with increased mortality. For IL-10, the stromal expression was highest in the primary tumors (P < 0.001), whereas in the metastases the expression was highest in tumor cell areas (P < 0.001). High IL-10 expression in tumor- and stromal cell areas of primary tumors predicted mortality. Ki67 was higher expressed in tumor stromal areas of the metastases, and in tumor cell areas of the primary tumors (P < 0.001). Ki67 expression in tumor cell areas and stromal areas of the metastases was independently associated with breast cancer mortality.

Conclusions

Stromal expression of COX-2, TGF-beta and Ki67 may facilitate tumor progression in breast cancer.     

ASPM-associated stem cell proliferation is involved in malignant progression of gliomas and constitutes an attractive therapeutic target

Background

ASPM (Abnormal Spindle-like Microcephaly associated) over-expression was recently implicated in the development of malignant gliomas.

Results

To better characterize the involvement of ASPM in gliomas, we investigated the mRNA expression in 175 samples, including 8 WHO Grade II, 75 WHO Grade III and 92 WHO Grade IV tumors. Aspm expression was strongly correlated with tumor grade and increased at recurrence when compared to the initial lesion, whatever the initial grade of the primary tumor. ASPM expression also increased over serial passages in gliomaspheres in vitro and in mouse xenografts in vivo. Lentivirus-mediated shRNA silencing of ASPM resulted in dramatic proliferation arrest and cell death in two different gliomasphere models.

Conclusion

These data suggest that ASPM is involved in the malignant progression of gliomas, possibly through expansion of a cancer stem cell compartment, and is an attractive therapeutic target in glioblastoma multiforme.     

Transcriptional profiling of Actinobacillus pleuropneumoniae under iron-restricted conditions

Background

Toll-like receptors (TLRs) play a central role in innate immunity. TLRs are membrane glycoproteins and contain leucine rich repeat (LRR) motif in the ectodomain. TLRs recognize and respond to molecules such as lipopolysaccharide, peptidoglycan, flagellin, and RNA from bacteria or viruses. The LRR domains in TLRs have been inferred to be responsible for molecular recognition. All LRRs include the highly conserved segment, LxxLxLxxNxL, in which "L" is Leu, Ile, Val, or Phe and "N" is Asn, Thr, Ser, or Cys and "x" is any amino acid. There are seven classes of LRRs including "typical" ("T") and "bacterial" ("S"). All known domain structures adopt an arc or horseshoe shape. Vertebrate TLRs form six major families. The repeat numbers of LRRs and their "phasing" in TLRs differ with isoforms and species; they are aligned differently in various databases. We identified and aligned LRRs in TLRs by a new method described here.

Results

The new method utilizes known LRR structures to recognize and align new LRR motifs in TLRs and incorporates multiple sequence alignments and secondary structure predictions. TLRs from thirty-four vertebrate were analyzed. The repeat numbers of the LRRs ranges from 16 to 28. The LRRs found in TLRs frequently consists of LxxLxLxxNxLxxLxxxxF/LxxLxx ("T") and sometimes short motifs including LxxLxLxxNxLxxLPx(x)LPxx ("S"). The TLR7 family (TLR7, TLR8, and TLR9) contain 27 LRRs. The LRRs at the N-terminal part have a super-motif of STT with about 80 residues. The super-repeat is represented by STTSTTSTT or _TTSTTSTT. The LRRs in TLRs form one or two horseshoe domains and are mostly flanked by two cysteine clusters including two or four cysteine residue.

Conclusion

Each of the six major TLR families is characterized by their constituent LRR motifs, their repeat numbers, and their patterns of cysteine clusters. The central parts of the TLR1 and TLR7 families and of TLR4 have more irregular or longer LRR motifs. These central parts are inferred to play a key role in the structure and/or function of their TLRs. Furthermore, the super-repeat in the TLR7 family suggests strongly that "bacterial" and "typical" LRRs evolved from a common precursor.     

Diffuse large B-cell lymphoma presenting with neurolymphomatosis and intravascular lymphoma: a unique autopsy case with diverse neurological symptoms

Background

The diagnosis of uterine smooth muscle tumors depends on a combination of microscopic features. However, a small number of these tumors still pose difficult diagnostic challenges.

Aim

To investigate progesterone receptor (PR) and p53 expression in leiomyomas (LMs), atypical leiomyomas (ALMs), smooth muscle tumors of uncertain malignant potential (STUMP), and leiomyosarcomas (LMSs) and to evaluate the potential utility of the selected immunohistochemical markers in differentiating these tumors.

Materials and methods

Immunohistochemical expression of PR and p53 was investigated in 41 uterine smooth muscle tumors comprising: 15 LMS, 4 STUMP, 6 ALM and 16 LM. Quantitative evaluation of PR and p53 expression was graded on a scale from 0 to 3+.

Results

Leiomyosarcomas showed reduced PR expression. All LMs as well as ALMs and STUMP were stained intensely for PR. Conversely, LMS was strongly stained with p53, while the three non-sarcomatous groups (STUMP, ALM, LM) were either entirely negative or weakly stained for p53. Regarding both PR and p53 expression, the difference between the LMS group and the three non-sarcomatous groups was highly significant (p < 0.001). Combined high PR - low p53 expression was seen in all the 26 examined cases of the non-sarcomatous group including the STUMP cases and none of the LMS cases. Therefore, it represents a "benign" profile with 100% specificity in diagnosis of a non-sarcomatous tumor.

Conclusion

Immunohistochemistry for PR and p53 is valuable as an adjunct tool to morphological assessment of problematic uterine smooth muscle tumors.     

The Glycolytic Inhibitor 2-Deoxyglucose Activates Multiple Prosurvival Pathways through IGF1R

Recent molecular studies indicate that aerobic glycolysis plays an important role in tumorigenesis and is a valid target for cancer therapy. Although 2-deoxyglucose (2-DG) is well characterized as a glycolytic inhibitor, we recently discovered that it activates a prosurvival oncoprotein, AKT, through PI3K. In this study, we discovered that 2-DG treatments disrupted the binding between insulin-like growth factor 1 (IGF-1) and IGF-binding protein 3 (IGFBP3) so that the free form of IGF-1 could be released from the IGF-1·IGFBP3 complex to activate IGF-1 receptor (IGF1R) signaling. Because IGF1R signaling is involved, PI3K/AKT constitutes only one of the prosurvival pathways that are activated by 2-DG treatment; we validated that MEK-ERK signaling was also induced in an IGF1R-dependent manner in some cancer cell lines. Furthermore, our phospho-specific antibody microarray analysis indicated that 2-DG up-regulated the phosphorylation of 64 sites within various signaling pathways in H460 cells. Chemical inhibition of IGF1R reduced 57 of these up-regulations. These data suggest that 2-DG-induced activation of many survival pathways can be jointly attenuated through IGF1R inhibition. Our in vitro analysis demonstrated that treatment with a combination of subtoxic doses of 2-DG and the IGF1R inhibitor II reduced cancer cell proliferation 90% and promoted significant apoptosis.Cancer cells display high rates of aerobic glycolysis in comparison with their nontransformed counterparts (i.e. the Warburg effect (1)). Whether increased aerobic glycolysis drives tumor formation or merely represents a byproduct of oncogenic transformation has been a subject of controversy. Two recent studies demonstrated that the Warburg effect can be reversed in some cancer cells by either the depletion of lactate dehydrogenenase A or switching pyruvate kinase expression from M2 to M1 isoform (2, 3). Interestingly, the reversal of the Warburg effect correlates with a reduction in the ability of the isogenic cancer cells to form tumors in nude mouse xenografts. Viewed in combination, these observations appeared to indicate that tumor cells preferentially use glucose for purposes other than oxidative phosphorylation and that aerobic glycolysis is a valid target for cancer therapeutics.Targeting glycolysis for cancer treatment has been explored previously as a therapeutic approach (4, 5). Of all the glycolysis inhibitors that were evaluated, 2-deoxyglucose (2-DG)³ is the one that has been best characterized in animal model studies and human clinical trials (6–8). It is converted by hexokinase to phosphorylated 2-DG, which becomes trapped inside the cell and inhibits hexokinase (9). As a direct consequence of 2-DG treatment, intracellular ATP is depleted (10, 11), which ultimately suppresses cell proliferation in vitro (12, 13). Nonetheless, the implementation of 2-DG as an anticancer agent in vivo has been a disappointment. Whereas 2-DG suppresses cell growth in vitro, studies using xenografts indicate that 2-DG treatment, when provided as a single agent, does not inhibit tumor growth (6).Because 2-DG is a small molecule, we suspected that it activates other signaling pathways and decided to evaluate its off-target effects. Our initial findings indicated that 2-DG activates AKT function through phosphatidylinositol 3-kinase (PI3K) and is independent of glycolysis or mTOR inhibition. Thus, the inhibitory effect on growth produced by 2-DG-mediated glycolysis inhibition may be partial offset by the fact there is also 2-DG-induced AKT activation (14). In the current study, we used a phospho-specific antibody array to identify IGF1R as the upstream receptor tyrosine kinase that is responsible for the activation of AKT signaling. Using recombinant IGF-1 and IGFBP3 proteins, we discovered that the inhibition of IGF-1 by IGFBP3 is disrupted in the presence of 2-DG. As 2-DG treatment activates IGF-1 signaling, we evaluated other prosurvival signaling pathways such as ERK signaling, which was also activated by 2-DG treatment in some cancer cell lines. Lastly, we tested to see whether an inhibitor of IGF1R would interfere with the prosurvival pathways and increase apoptosis if given in combination with 2-DG.     

In vivo analysis of fracture toughness of thyroid gland tumors

Background

Human solid tumors that are hard or firm on physical palpation are likely to be cancerous, a clinical maxim that has been successfully applied to cancer screening programs, such as breast self-examination. However, the biological relevance or prognostic significance of tumor hardness remains poorly understood. Here we present a fracture mechanics based in vivo approach for characterizing the fracture toughness of biological tissue of human thyroid gland tumors.

Methods

In a prospective study, 609 solid thyroid gland tumors were percutaneously probed using standard 25 gauge fine needles, their tissue toughness ranked on the basis of the nature and strength of the haptic force feedback cues, and subjected to standard fine needle biopsy. The tumors' toughness rankings and final cytological diagnoses were combined and analyzed. The interpreting cytopathologist was blinded to the tumors' toughness rankings.

Results

Our data showed that cancerous and noncancerous tumors displayed remarkable haptically distinguishable differences in their material toughness.

Conclusion

The qualitative method described here, though subject to some operator bias, identifies a previously unreported in vivo approach to classify fracture toughness of a solid tumor that can be correlated with malignancy, and paves the way for the development of a mechanical device that can accurately quantify the tissue toughness of a human tumor.     

Waveguide model of the hearing aid earmold system

Background

Epstein-Barr virus (EBV) is associated to the etio-pathogenesis of an increasing number of tumors. Detection of EBV in pathology samples is relevant since its high prevalence in some cancers makes the virus a promising target of specific therapies. RNA in situ hybridization (RISH) is the standard diagnostic procedure, while polymerase chain reaction (PCR)-based methods are used for strain (EBV type-1 or 2) distinction. We performed a systematic comparison between RISH and PCR for EBV detection, in a group of childhood B-cell Non-Hodgkin lymphomas (NHL), aiming to validate PCR as a first, rapid method for the diagnosis of EBV-associated B-cell NHL.

Methods

EBV infection was investigated in formalin fixed paraffin-embedded tumor samples of 41 children with B-cell NHL, including 35 Burkitt's lymphoma (BL), from Rio de Janeiro, Brazil, by in situ hybridization of EBV-encoded small RNA (EBER-RISH) and PCR assays based on EBNA2 amplification.

Results

EBV genomes were detected in 68% of all NHL. Type 1 and 2 accounted for 80% and 20% of EBV infection, respectively. PCR and RISH were highly concordant (95%), as well as single- and nested-PCR results, allowing the use of a single PCR round for diagnostic purposes. PCR assays showed a sensitivity and specificity of 96% and 100%, respectively, with a detection level of 1 EBV genome in 5,000–10,000 EBV-negative cells, excluding the possibility of detecting low-number EBV-bearing memory cells.

Conclusion

We describe adequate PCR conditions with similar sensitivity and reliability to RISH, to be used for EBV diagnostic screening in high grade B-NHL, in "at risk" geographic regions.     

Antiangiogenic activity of 2-deoxy-D-glucose

Background

During tumor angiogenesis, endothelial cells (ECs) are engaged in a number of energy consuming biological processes, such as proliferation, migration, and capillary formation. Since glucose uptake and metabolism are increased to meet this energy need, the effects of the glycolytic inhibitor 2-deoxy-D-glucose (2-DG) on in vitro and in vivo angiogenesis were investigated.

Methodology/Principal Findings

In cell culture, 2-DG inhibited EC growth, induced cytotoxicity, blocked migration, and inhibited actively forming but not established endothelial capillaries. Surprisingly, 2-DG was a better inhibitor of these EC properties than two more efficacious glycolytic inhibitors, 2-fluorodeoxy-D-glucose and oxamate. As an alternative to a glycolytic inhibitory mechanism, we considered 2-DG''s ability to interfere with endothelial N-linked glycosylation. 2-DG''s effects were reversed by mannose, an N-linked glycosylation precursor, and at relevant concentrations 2-DG also inhibited synthesis of the lipid linked oligosaccharide (LLO) N-glycosylation donor in a mannose-reversible manner. Inhibition of LLO synthesis activated the unfolded protein response (UPR), which resulted in induction of GADD153/CHOP and EC apoptosis (TUNEL assay). Thus, 2-DG''s effects on ECs appeared primarily due to inhibition of LLOs synthesis, not glycolysis. 2-DG was then evaluated in two mouse models, inhibiting angiogenesis in both the matrigel plug assay and the LH_BETAT_AG transgenic retinoblastoma model.

Conclusions/Significance

In conclusion, 2-DG inhibits endothelial cell angiogenesis in vitro and in vivo, at concentrations below those affecting tumor cells directly, most likely by interfering with N-linked glycosylation rather than glycolysis. Our data underscore the importance of glucose metabolism on neovascularization, and demonstrate a novel approach for anti-angiogenic strategies.     

Occurrence of cancer at multiple sites: Towards distinguishing multigenesis from metastasis

Background

Occurrence of tumors at multiple sites is a hallmark of malignant cancers and contributes to the high mortality of cancers. The formation of multi-site cancers (MSCs) has conventionally been regarded as a result of hematogenous metastasis. However, some MSCs may appear as unusual in the sense of vascular dissemination pattern and therefore be explained by alternative metastasis models or even by non-metastatic independent formation mechanisms.

Results

Through literature review and incorporation of recent advance in understanding aging and development, we identified two alternative mechanisms for the independent formation of MSCs: 1) formation of separate tumors from cancer-initiating cells (CICs) mutated at an early stage of development and then diverging as to their physical locations upon further development, 2) formation of separate tumors from different CICs that contain mutations in some convergent ways. Either of these processes does not require long-distance migration and/or vascular dissemination of cancer cells from a primary site to a secondary site. Thus, we classify the formation of these MSCs from indigenous CICs (iCICs) into a new mechanistic category of tumor formation – multigenesis.

Conclusion

A multigenesis view on multi-site cancer (MSCs) may offer explanations for some "unusual metastasis" and has important implications for designing expanded strategies for the diagnosis and treatment of cancers.

Reviewers

This article was reviewed by Carlo C. Maley nominated by Laura F. Landweber and Razvan T. Radulescu nominated by David R. Kaplan. For the full reviews, please go to the Reviewers' comments section.     

Morphology and head morphometric characters of sperm in Thai native crossbred stallions

Background

In women with breast cancer submitted to neoadjuvant chemotherapy based in doxorubicin, tumor expression of groups of three genes (PRSS11, MTSS1, CLPTM1 and PRSS11, MTSS1, SMYD2) have classified them as responsive or resistant. We have investigated whether expression of these trios of genes could predict mammary carcinoma response in dogs and whether tumor slices, which maintain epithelial-mesenchymal interactions, could be used to evaluate drug response in vitro.

Methods

Tumors from 38 dogs were sliced and cultured with or without doxorubicin 1 μM for 24 h. Tumor cells were counted by two observers to establish a percentage variation in cell number, between slices. Based on these results, a reduction in cell number between treated and control samples ≥ 21.7%, arbitrarily classified samples, as drug responsive. Tumor expression of PRSS11, MTSS1, CLPTM1 and SMYD2, was evaluated by real time PCR. Relative expression results were then transformed to their natural logarithm values, which were spatially disposed according to the expression of trios of genes, comprising PRSS11, MTSS1, CLPTM1 and PRSS11, MTSS1, SMYD2. Fisher linear discrimination test was used to generate a separation plane between responsive and non-responsive tumors.

Results

Culture of tumor slices for 24 h was feasible. Nine samples were considered responsive and 29 non-responsive to doxorubicin, considering the pre-established cut-off value of cell number reduction ≥ 21.7%, between doxorubicin treated and control samples. Relative gene expression was evaluated and tumor samples were then spatially distributed according to the expression of the trios of genes: PRSS11, MTSS1, CLPTM1 and PRSS11, MTSS1, SMYD2. A separation plane was generated. However, no clear separation between responsive and non-responsive samples could be observed.

Conclusion

Three-dimensional distribution of samples according to the expression of the trios of genes PRSS11, MTSS1, CLPTM1 and PRSS11, MTSS1, SMYD2 could not predict doxorubicin in vitro responsiveness. Short term culture of mammary gland cancer slices may be an interesting model to evaluate chemotherapy activity.     

An inhibitor of K+ channels modulates human endometrial tumor-initiating cells

Background

Many potassium ion (K⁺) channels function as oncogenes to sustain growth of solid tumors, but their role in cancer progression is not well understood. Emerging evidence suggests that the early progenitor cancer cell subpopulation, termed tumor initiating cells (TIC), are critical to cancer progression.

Results

A non-selective antagonist of multiple types of K⁺ channels, tetraethylammonium (TEA), was found to suppress colony formation in endometrial cancer cells via inhibition of putative TIC. The data also indicated that withdrawal of TEA results in a significant enhancement of tumorigenesis. When the TIC-enriched subpopulation was isolated from the endometrial cancer cells, TEA was also found to inhibit growth in vitro.

Conclusions

These studies suggest that the activity of potassium channels significantly contributes to the progression of endometrial tumors, and the antagonists of potassium channels are candidate anti-cancer drugs to specifically target tumor initiating cells in endometrial cancer therapy.     

Rapid diagnostic tests as a source of DNA for Plasmodium species-specific real-time PCR

Background

To overcome the problem of increasing drug resistance, traditional medicines are an important source for potential new anti-malarials. Caesalpinia pluviosa, commonly named "sibipiruna", originates from Brazil and possess multiple therapeutic properties, including anti-malarial activity.

Methods

Crude extract (CE) was obtained from stem bark by purification using different solvents, resulting in seven fractions. An MTT assay was performed to evaluate cytotoxicity in MCF-7 cells. The CE and its fractions were tested in vitro against chloroquine-sensitive (3D7) and -resistant (S20) strains of Plasmodium falciparum and in vivo in Plasmodium chabaudi-infected mice. In vitro interaction with artesunate and the active C. pluviosa fractions was assessed, and mass spectrometry analyses were conducted.

Results

At non-toxic concentrations, the 100% ethanolic (F4) and 50% methanolic (F5) fractions possessed significant anti-malarial activity against both 3D7 and S20 strains. Drug interaction assays with artesunate showed a synergistic interaction with the F4. Four days of treatment with this fraction significantly inhibited parasitaemia in mice in a dose-dependent manner. Mass spectrometry analyses revealed the presence of an ion corresponding to m/z 303.0450, suggesting the presence of quercetin. However, a second set of analyses, with a quercetin standard, showed distinct ions of m/z 137 and 153.

Conclusions

The findings show that the F4 fraction of C. pluviosa exhibits anti-malarial activity in vitro at non-toxic concentrations, which was potentiated in the presence of artesunate. Moreover, this anti-malarial activity was also sustained in vivo after treatment of infected mice. Finally, mass spectrometry analyses suggest that a new compound, most likely an isomer of quercetin, is responsible for the anti-malarial activity of the F4.     

Engineering of N. benthamiana L. plants for production of N-acetylgalactosamine-glycosylated proteins - towards development of a plant-based platform for production of protein therapeutics with mucin type O-glycosylation

Background

Mucin type O-glycosylation is one of the most common types of post-translational modifications that impacts stability and biological functions of many mammalian proteins. A large family of UDP-GalNAc polypeptide:N-acetyl-α-galactosaminyltransferases (GalNAc-Ts) catalyzes the first step of mucin type O-glycosylation by transferring GalNAc to serine and/or threonine residues of acceptor polypeptides. Plants do not have the enzyme machinery to perform this process, thus restricting their use as bioreactors for production of recombinant therapeutic proteins.

Results

The present study demonstrates that an isoform of the human GalNAc-Ts family, GalNAc-T2, retains its localization and functionality upon expression in N. benthamiana L. plants. The recombinant enzyme resides in the Golgi as evidenced by the fluorescence distribution pattern of the GalNAc-T2:GFP fusion and alteration of the fluorescence signature upon treatment with Brefeldin A. A GalNAc-T2-specific acceptor peptide, the 113-136 aa fragment of chorionic gonadotropin β-subunit, is glycosylated in vitro by the plant-produced enzyme at the "native" GalNAc attachment sites, Ser-121 and Ser-127. Ectopic expression of GalNAc-T2 is sufficient to "arm" tobacco cells with the ability to perform GalNAc-glycosylation, as evidenced by the attachment of GalNAc to Thr-119 of the endogenous enzyme endochitinase. However, glycosylation of highly expressed recombinant glycoproteins, like magnICON-expressed E. coli enterotoxin B subunit: H. sapiens mucin 1 tandem repeat-derived peptide fusion protein (LTBMUC1), is limited by the low endogenous UDP-GalNAc substrate pool and the insufficient translocation of UDP-GalNAc to the Golgi lumen. Further genetic engineering of the GalNAc-T2 plants by co-expressing Y. enterocolitica UDP-GlcNAc 4-epimerase gene and C. elegans UDP-GlcNAc/UDP-GalNAc transporter gene overcomes these limitations as indicated by the expression of the model LTBMUC1 protein exclusively as a glycoform.

Conclusion

Plant bioreactors can be engineered that are capable of producing Tn antigen-containing recombinant therapeutics.     

Is type 2 diabetes mellitus a vascular disease (atheroscleropathy) with hyperglycemia a late manifestation? The role of NOS, NO, and redox stress.

Background

The impressive correlation between cardiovascular disease and glucose metabolism alterations has raised the likelihood that atherosclerosis and type 2 diabetes may share common antecedents. Inflammation is emerging as a conceivable etiologic mechanism for both. Interleukins are regulatory proteins with ability to accelerate or inhibit inflammatory processes.

Presentation of the hypothesis

A novel interleukins classification is described, based on their role in diabetes and atherosclerosis, hypothesizing that each interleukin (IL) acts on both diseases in the same direction – regardless if harmful, favorable or neutral.

Testing the hypothesis

The 29 known interleukins were clustered into three groups: noxious (the "bad", 8 members), comprising IL-1, IL-2, IL-6, IL-7, IL-8, IL-15, IL-17 and IL-18; protective (the "good", 5 members), comprising IL-4, IL-10, IL-11, IL-12 and IL-13; and "aloof", comprising IL-5, IL-9, IL-14, IL-16 and IL-19 through IL-29 (15 members). Each group presented converging effects on both diseases. IL-3 was reluctant to clustering.

Implications

These observations imply that 1) favorable effects of a given IL on either diabetes or atherosclerosis predicts similar effects on the other; 2) equally, harmful IL effects on one disease can be extrapolated to the other; and 3) absence of influence of a given IL on one of these diseases forecasts lack of effects on the other. These facts further support the unifying etiologic theory of both ailments, emphasizing the importance of a cardiovascular diabetologic approach to interleukins for future research. Pharmacologic targeting of these cytokines might provide an effective means to simultaneously control both atherosclerosis and diabetes.     

Biological processes, properties and molecular wiring diagrams of candidate low-penetrance breast cancer susceptibility genes

Background

The process of malignant transformation, progression and metastasis of melanoma is poorly understood. Gene expression profiling of human cancer has allowed for a unique insight into the genes that are involved in these processes. Thus, we have attempted to utilize this approach through the analysis of a series of primary, non-metastatic cutaneous tumors and metastatic melanoma samples.

Methods

We have utilized gene microarray analysis and a variety of molecular techniques to compare 40 metastatic melanoma (MM) samples, composed of 22 bulky, macroscopic (replaced) lymph node metastases, 16 subcutaneous and 2 distant metastases (adrenal and brain), to 42 primary cutaneous cancers, comprised of 16 melanoma, 11 squamous cell, 15 basal cell skin cancers. A Human Genome U133 Plus 2.0 array from Affymetrix, Inc. was utilized for each sample. A variety of statistical software, including the Affymetrix MAS 5.0 analysis software, was utilized to compare primary cancers to metastatic melanomas. Separate analyses were performed to directly compare only primary melanoma to metastatic melanoma samples. The expression levels of putative oncogenes and tumor suppressor genes were analyzed by semi- and real-time quantitative RT-PCR (qPCR) and Western blot analysis was performed on select genes.

Results

We find that primary basal cell carcinomas, squamous cell carcinomas and thin melanomas express dramatically higher levels of many genes, including SPRR1A/B, KRT16/17, CD24, LOR, GATA3, MUC15, and TMPRSS4, than metastatic melanoma. In contrast, the metastatic melanomas express higher levels of genes such as MAGE, GPR19, BCL2A1, MMP14, SOX5, BUB1, RGS20, and more. The transition from non-metastatic expression levels to metastatic expression levels occurs as melanoma tumors thicken. We further evaluated primary melanomas of varying Breslow's tumor thickness to determine that the transition in expression occurs at different thicknesses for different genes suggesting that the "transition zone" represents a critical time for the emergence of the metastatic phenotype. Several putative tumor oncogenes (SPP-1, MITF, CITED-1, GDF-15, c-Met, HOX loci) and suppressor genes (PITX-1, CST-6, PDGFRL, DSC-3, POU2F3, CLCA2, ST7L), were identified and validated by quantitative PCR as changing expression during this transition period. These are strong candidates for genes involved in the progression or suppression of the metastatic phenotype.

Conclusion

The gene expression profiling of primary, non-metastatic cutaneous tumors and metastatic melanoma has resulted in the identification of several genes that may be centrally involved in the progression and metastatic potential of melanoma. This has very important implications as we continue to develop an improved understanding of the metastatic process, allowing us to identify specific genes for prognostic markers and possibly for targeted therapeutic approaches.