Weak Protein Expression Research Articles

Generation of embryonic stem cells derived from the inner cell mass of blastocysts of outbred ICR mice

ABSTRACT Embryonic stem cells (ESCs) derived from outbred mice which share several genetic characteristics similar to humans have been requested for developing stem cell-based bioengineering techniques directly applicable to humans. Here, we report the generation of ESCs derived from the inner cell mass of blastocysts retrieved from 9-week-old female outbred ICR mice mated with 9-week-old male outbred ICR mice (ICRESCs). Similar to those from 129/Ola mouse blastocysts (E14ESCs), the established ICRESCs showed inherent characteristics of ESCs except for partial and weak protein expression and activity of alkaline phosphatase. Moreover, ICRESCs were not originated from embryonic germ cells or pluripotent cells that may co-exist in outbred ICR strain-derived mouse embryonic fibroblasts (ICRMEFs) used for deriving colonies from inner cell mass of outbred ICR mouse blastocysts. Furthermore, instead of outbred ICRMEFs, hybrid B6CBAF1MEFs as feeder cells could sufficiently support in vitro maintenance of ICRESC self-renewal. Additionally, ICRESC-specific characteristics (self-renewal, pluripotency, and chromosomal normality) were observed in ICRESCs cultured for 40th subpassages (164 days) on B6CBAF1MEFs without any alterations. These results confirmed the successful establishment of ESCs derived from outbred ICR mice, and indicated that self-renewal and pluripotency of the established ICRESCs could be maintained on B6CBAF1MEFs in culture.

Correlation Between MYCN Gene Status and MYCN Protein Expression in Neuroblastoma: A Pilot Study To Propose the Use of MYCN Immunohistochemistry in Limited-Resource Areas

PURPOSEThe most significant adverse risk factor for neuroblastoma (NB) is MYCN gene amplification, which strongly associates with high-risk disease. Fluorescent in situ hybridization (FISH) is considered the best method to evaluate MYCN gene status. However, it requires a laboratory that can perform highly complex testing, specialized personnel, and costly reagents. Herein, we aimed to investigate the feasibility of using immunohistochemistry (IHC) to detect MYCN protein expression in lieu of FISH, a strategy potentially useful in areas with limited resources.METHODSA pilot cohort of 78 patients with NB, including 34 of Middle Eastern descent (MED) who had a higher prevalence of MYCN gene amplification (44.11%) and 44 of North American descent (NAD), nine (20.45%) of whom had MYCN amplification, was evaluated with IHC for MYCN protein. Correlations of FISH results and protein expression are presented.RESULTSA positive correlation between MYCN gene amplification and protein expression by IHC was seen in 22 (91.66%) of the 24 MYCN-amplified NB cases—14 (93.33%) of 15 patients of MED and eight (88.88%) of nine patients of NAD. Agreement between negative FISH and negative IHC results was noted in 18 (94.73%) patients of MED and 34 (97.14%) patients of NAD. Two cases had weak protein expression but no gene amplification (MED: n = 1; 5.0%; NAD: n = 1; 2.9%).CONCLUSIONAn excellent overall correlation between MYCN gene status by FISH and MYCN protein expression by IHC was confirmed. MYCN IHC in NB with reflexing to FISH in equivocal cases is potentially useful in a limited-resource setting. Evaluation of effectiveness using a larger cohort and optimization to perform MYCN IHC manually is needed.

Astemizole inhibits cell proliferation in human prostate tumorigenic cells expressing ether à-go-go-1 potassium channels.

Prostate cancer (PC) is the main cause of cancer mortality in men worldwide. Therefore, novel treatments for PC are needed. Ether à-go-go-1 (Eag1) potassium channels display oncogenic properties, and have been suggested as early tumor markers and therapeutic targets for different cancers. These channels are overexpressed in many human tumors including PC. Astemizole targets several molecules involved in cancer including Eag1 channels, histamine receptors and ABC transporters. Here we studied Eag1 mRNA expression and protein levels in the non-tumorigenic and non-invasive human prostate RWPE-1 cell line, and in the tumorigenic and highly invasive human prostate WPE1-NB26 cell lines. The effect of astemizole on cell proliferation and apoptosis was also studied. The human prostate cell lines RWPE-1 and WPE1-NB26 were cultured following the provider´s instructions. Eag1 mRNA expression and protein levels were studied by real time RT-PCR and immunocytochemistry, respectively. Cell proliferation and apoptosis were studied by a fluorescence AlamarBlue® assay and flow cytometry, respectively. No difference in Eag1 mRNA expression was observed between the cell lines. However, high Eag1 protein levels were observed in the invasive WPE1-NB26 cells, in contrast to the weak protein expression in RWPE-1 cells. Accordingly, astemizole decreased cell proliferation at nanomolar concentrations only in the invasive WPE1-NB26 cells. Our results suggest that astemizole may have clinical relevance for prostate cancer treatment in patients with high Eag1 protein levels.

Astemizole inhibits cell proliferation in human prostate tumorigenic cells expressing ether í -go-go-1 potassium channels

Prostate cancer (PC) is the main cause of cancer mortality in men worldwide. Therefore, novel treatments for PC are needed. Ether í -go-go-1 (Eag1) potassium channels display oncogenic properties, and have been suggested as early tumor markers and therapeutic targets for different cancers. These channels are overexpressed in many human tumors including PC. Astemizole targets several molecules involved in cancer including Eag1 channels, histamine receptors and ABC transporters. Here we studied Eag1 mRNA expression and protein levels in the non-tumorigenic and non-invasive human prostate RWPE-1 cell line, and in the tumorigenic and highly invasive human prostate WPE1-NB26 cell lines. The effect of astemizole on cell proliferation and apoptosis was also studied. The human prostate cell lines RWPE-1 and WPE1-NB26 were cultured following the provider´s instructions. Eag1 mRNA expression and protein levels were studied by real time RT-PCR and immunocytochemistry, respectively. Cell proliferation and apoptosis were studied by a fluorescence AlamarBlue® assay and flow cytometry, respectively. No difference in Eag1 mRNA expression was observed between the cell lines. However, high Eag1 protein levels were observed in the invasive WPE1-NB26 cells, in contrast to the weak protein expression in RWPE-1 cells. Accordingly, astemizole decreased cell proliferation at nanomolar concentrations only in the invasive WPE1-NB26 cells. Our results suggest that astemizole may have clinical relevance for prostate cancer treatment in patients with high Eag1 protein levels.

Effect of PFT-α on apoptosis of spermatogenic cells caused by enorchia

To determine the molecular mechanism of germ cell apoptosis via investigating the effect of PFT-α on the expression of p53 and bcl-2/bax during experimental cryptorchid cell apoptosis. Male Sprague-Dawley rats were assigned into 4 groups: a sham-operated group, a cryptorchid group, a cryptorchid+p53 inhibitor (p53 inhibitor-alpha, PFT-α) group, and a cryptorchid+dissolvent of PFT-α [dimethyl sulphoxide (DMSO)] group. Unilateral cryptorchidism was surgically induced in the rats of the cryptorchid group, PFT-α group, and cryptorchid+dissolvent of PFT-α group. The rats in the PFT-α group and cryptorchid+dissolvent of PFT-α group were intra-peritoneally injected PFT-α and dissolvent of PFT-α, respectively, once a day. The rats were killed on the 7th day after the surgery. The morphology of spermatogenic epithelium at the side of surgery in the rats was observed under light microscope. The apoptosis of spermatogenic cells in the unilateral cryptorchidism was evaluated by TUNEL and flow cytometry (FCM). The protein expression levels of p53, bcl-2, and Bax were detected by Western blot and immunohistochemical assay in turn. Compared with the cryptorchid groups and the cryptorchid+dissolvent of PFT-α group, the seminiferous epithelium of the cryptorchid+p53 inhibitor group appeared orderly, with thicker cell layers and lower apoptosis index, weak protein expression level of p53/Bax and strong protein expression level of bcl-2. PFT-α inhibits the germ cell apoptosis caused by the experimental cryptorchidism via increasing the expression of bcl-2 and decreasing the expression of p53 and bax.

Proteomic identification of Reticulocalbin 1 as potential tumor marker in renal cell carcinoma

Renal cell carcinoma (RCC) biomarkers are necessary for diagnosis and prognosis. They serve to monitor therapy response and follow-up, as drug targets, and therapy predictors in personalized treatments. Proteomics is a suitable method for biomarker discovery. Here we investigate differential protein expression in RCC, and we evaluate Reticulocalbin 1 (RCN1) use as a new potential marker. Neoplastic and healthy tissue samples were collected from 24 RCC patients during radical nephrectomy. Seven specimens were firstly processed by proteomic analysis (2-DE and MALDI-TOF) and 18 differentially expressed proteins from neoplastic and healthy renal tissues were identified. Among them, RCN1 was over-expressed in all cancer specimens analyzed by proteomics. Consequently RCN1 use as a potential marker was further evaluated in all 24 donors. RCN1 expression was verified by Western blotting (WB) and immunohistochemistry (IHC). WB analysis confirmed RCN1 over-expression in 21 out of 24 tumor specimens, whereas IHC displayed focal or diffuse expression of RCN1 in all 24 RCC tissues. Thus RCN1 appears as a potential marker for clinical approaches. A larger histopathological trial will clarify the prognostic value of RCN1 in RCC. The present work aimed at finding new biomarkers for RCC - a life-threatening disease characterized by high incidence in Western countries - by performing differential proteomic analysis of neoplastic and normal renal tissues obtained from a small cohort of RCC patients. Some of the identified proteins have been previously associated to renal cancer however data confirming the possible use of these proteins in clinical practice are not available to date. By IHC we demonstrated that RCN1 could be easily employed in clinical practice, confirming RCN1 over-expression in RCC tissues of all examined patients, and weak protein expression in healthy renal tissues only in correspondence to the renal tubule section. These data indicate a promising role of RCN1 as a possible marker in RCC and indicate the proximal convoluted renal tubule as a putative origin point for RCC. Since IHC staining displayed different grades of intensity in tested tissues, we hypothesized that RCN1 could also be employed as a prognostic marker or as a response predictor for RCC-targeted therapy. To test such a hypothesis, a larger retrospective trial on paraffin-embedded tissues obtained from radical or partial nephrectomy of RCC patients is planned to be performed by our group.

Expression of peroxiredoxin I in the rats exposed to silica

To evaluate the change in protein expression of peroxiredoxin I (Prx I) during pulmonary fibrosis among rats exposed to silica dust and to investigate the role of Prx I in pulmonary fibrosis. Ninety male Wistar rats were randomly divided into control group (n = 60) and experimental group (n = 30). The control group received intratracheal perfusion of saline (1 ml), while the experimental group received intratracheal perfusion of suspension of silica dust (50 mg/ml) to establish a rat model of silicosis. At 1, 2, 3, 4, 6, or 8 weeks after treatment, 10 rats in control group and 5 rats in experimental group were sacrificed. The lung tissues were collected for conventional pathological observation. The protein expression of Prx I at each time point was measured by immunohistochemistry and Western blot. Among the rats exposed to silica dust, Prx I was seen in the form of brown particles that were mainly distributed in the alveolar septa and the cytoplasm of alveolar epithelial cells, macrophages, vascular endothelial cells, and smooth muscle cells around the blood vessels and tracheae. The control group showed weak protein expression of Prx I, and the experimental group had significantly higher protein expression of Prx I than the control group at all time points (P < 0.05). In the experimental group, the protein expression of Prx I was upregulated significantly at 1 and 2 weeks and decreased at 3∼8 weeks. The change in protein expression of Prx I may be one of the important causes of the onset and development of pulmonary fibrosis in rats exposed to free silica.

Codon usage roles in human papillomavirus

Human papillomavirus (HPV) genomes, similar to other virus genomes, frequently have a G + C content significantly different from their host species. The HPV genomes show a strong codon usage bias to 18 codons, with 14 showing T at the third position amongst degenerately encoded amino acids. The codon usage pattern in HPV genome plays an important role, which regulates low or non-translational expression of the viral capsid genes and results in very weak protein expression of oncogenes in a wide range of mammalian cells. Codon modification has been proved to be a powerful technology to overcome the translational blockage and weak expression of both HPV capsid genes and oncogenes in different expression systems. Furthermore, keratinocytes are the host cells of HPV infection; the codon usage in HPV capsid genes matches available aminoacyl-tRNAs in differentiated keratinocytes to modulate their protein expression. HPV DNA vaccines with codon optimization have been shown to have higher immunogenicity and induce both strong cellular and humoral responses in animal models, which may be a promising form of therapeutic HPV vaccines.

Galectin-3 Expression is Ubiquitous in Tumors of the Sellar Region, Nervous System, and Mimics

Galectin-3 expression has been reported in spindle cell oncocytoma, certain pituitary adenoma subtypes, astrocytomas, oligodendrogliomas, and meningiomas. We evaluated galectin-3 protein expression by immunohistochemistry in 201 cases of a variety of nervous system and sellar tumors, as well as mRNA expression by reverse transcription-polymerase chain reaction in formalin-fixed paraffin-embedded tissue in a subset (20 cases). Immunohistochemical results were evaluated in a semiquantitative fashion on a 4-tiered scale (0 to 3). Strong (3+) immunoreactivity was seen in most of the cases (61%), followed by 2+(22%), and 1+(13%) staining. Only 4% of the lesions studied were immunonegative. Galectin-3 mRNA was present in 15 of the 18 cases (83%) in which reverse transcription-polymerase chain reaction was successful. Significant differences in protein expression were noted in the following 2 settings: specific meningioma subtypes (P=0.004, Fisher exact test) wherein clear cell meningioma demonstrated weak protein expression when compared with other meningioma variants. No significant difference was noted with respect to World Health Organization grade. Galectin-3 was also strongly expressed in benign nerve sheath tumors but only moderately expressed in malignant peripheral nerve sheath tumors (P=0.0009, Fisher exact test). Although galectin-3 positivity is a key feature of the immunophenotype of spindle cell oncocytoma, its consistent expression in other morphologically similar tumors (meningioma, pituicytoma, nerve sheath tumors, granular cell tumor, metastases) makes it of little use in the differential diagnosis of sellar region tumors, a setting in which it should be discouraged. Diagnostic uses of this marker may be limited to specific settings, including some meningioma subtypes and nerve sheath tumors.

Molecular basis of glutathione reductase deficiency in human blood cells

AbstractHereditary glutathione reductase (GR) deficiency was found in only 2 cases when testing more than 15 000 blood samples. We have investigated the blood cells of 2 patients (1a and 1b) in a previously described family suffering from favism and cataract and of a novel patient (2) presenting with severe neonatal jaundice. Red blood cells and leukocytes of the patients in family 1 did not contain any GR activity, and the GR protein was undetectable by Western blotting. Owing to a 2246-bp deletion in the patients' DNA, translated GR is expected to lack almost the complete dimerization domain, which results in unstable and inactive enzyme. The red blood cells from patient 2 did not exhibit GR activity either, but the patient's leukocytes contained some residual activity that correlated with a weak protein expression. Patient 2 was found to be a compound heterozygote, with a premature stop codon on one allele and a substitution of glycine 330, a highly conserved residue in the superfamily of NAD(P)H-dependent disulfide reductases, into alanine on the other allele. Studies on recombinant GR G330A revealed a drastically impaired thermostability of the protein. This is the first identification of mutations in the GR gene causing clinical GR deficiency.

Evaluation of DNA vaccination of spotted sand bass ( Paralabrax maculatofasciatus) with two major outer-membrane protein-encoding genes from Aeromonas veronii

Genes encoding two major outer membrane proteins (OMPs) of the bacterial pathogen Aeromonas veronii, Omp38 and Omp48, were used to construct DNA vaccines. The protective effect of such vaccines against motile aeromonad septicaemia was evaluated in spotted sand bass ( Paralabrax maculatofasciatus), an endemic species of the Mexican Northwest Pacific coast and a potential resource for the aquaculture industry. Weak protein expression, as determined by immunoblotting, was observed after transfection of eukaryotic cells with the DNA vaccines. Fish immunized with a single intramuscular injection of 20 μg of the omp38 and omp48 DNA vaccines showed slightly, but significantly elevated serum antibody levels 4 and 6 weeks after vaccination, compared to fish vaccinated with the control plasmid pcDNA3.1. Spotted sand bass vaccinated with the omp38 and omp48 DNA vaccines and challenged with A. veronii by intraperitoneal route recorded a relative percent survival (RPS) between 50 and 60%. Histopathological signs of motile aeromonad septicaemia were observed in around 40% of omp38 and omp48-vaccinated fish and 80% of pcDNA3.1-vaccinated control fish. The results indicate that P. maculatofasciatus vaccinated with a single dose of DNA plasmids encoding the major OMPs from A. veronii shows partial protection against infection and mortality by A. veronii experimental infection.

Numerical abnormalities of the Cyclin D 1 gene locus on chromosome 11q13 in non-melanoma skin cancer

Deregulation of the cell-cycle G1-restriction point control via abnormalities of Rb-pathway components is a frequent event in the formation of cancer. The aim of this study was to evaluate numerical aberrations of the Cyclin D 1 (CCND 1, PRAD1, bcl-1) gene locus at chromosome 11q13 in basal cell carcinomas (BCCs) and squamous cell carcinomas (SCCs) of the skin and to compare it with the Cyclin D 1 protein expression. Fluorescence in situ hybridization with DNA-probes specific for the Cyclin D 1 gene locus and the centromere of chromosome 11 as well as immunostaining for Cyclin D 1 protein was applied on 5 μm serial paraffin sections. Six of the 30 (20%) SCCs showed additional Cyclin D 1 gene copies and 2/30 (6.6%) cases had a loss of the Cyclin D 1 gene locus in relation to the centromere 11 number. In contrast, only one of the 14 BCCs (7%) showed one additional Cyclin D 1 gene copy in relation to the centromere 11 number. None of the BCCs demonstrated aneusomy for chromosome 11 in contrast to SCCs, where it was found in 21/30 (70%) cases. Twenty-six of the 30 (86.6%) cutaneous SCCs and 13/14 (93%) BCCs expressed Cyclin D 1 protein. All SCCs and the BCC with additional Cyclin D 1 gene copies showed positivity for Cyclin D 1 protein. Both SCCs with less Cyclin D 1 gene copies than centromere 11 signals showed a weak protein expression. Our findings suggest that numerical abnormalities of the Cyclin D 1 gene locus could result in an altered gene-dose effect, possibly leading to an aberrant expression in affected tumor cells. This might result in deregulation of cell cycle control, eventually leading to uncontrolled cell cycle progression.

Immunohistochemical study of components of the basement membrane in odontogenic cysts

The pattern of distribution and expression of laminin and type IV collagen was studied in ten radicular cysts, ten dentigerous cysts and ten odontogenic keratocysts, by means of the streptavidin-biotin method. The purpose of this study was to investigate changes in the distribution of components of the basement membrane in an attempt to contribute to the understanding of the differences, as to evolution and clinical behavior, between these cysts. The results revealed a weak and discontinuous linear staining, in odontogenic keratocysts, for both laminin and type IV collagen, while, in radicular cysts, staining was more intense and continuous. In dentigerous cysts, an intermediary pattern was observed, which was more similar to that observed in keratocysts. Our results suggest that, in view of the weak expression of proteins of the basement membrane observed in odontogenic keratocysts, modifications must take place in the interaction between the epithelium and the adjacent connective tissue, which could, in part, contribute to the pattern of more rapid growth exhibited by these cysts.

Expression of netrin-1 and netrin-1 receptor, DCC, in the rat olfactory nerve pathway during development and axonal regeneration

Netrin-1 is a bifunctional secreted protein that directs axon extension in various groups of developing axonal tracts. The transmembrane DCC (deleted in colorectal cancer) receptor is described as netrin-1 receptor and is involved in the attractive effects of netrin-1. In this study, we examined the spatio-temporal expression patterns of both netrin-1 and DCC in the rat olfactory system at different stages of development and during axonal regeneration following unilateral bulbectomy. High DCC expression was detected on the pioneer olfactory axons as they are extending toward the telencephalon. This expression was transient since from embryonic day 16 onwards, DCC was no longer detected along the olfactory nerve path. From embryonic day 14 until birth, DCC was also expressed within the mesenchyme surrounding the olfactory epithelium. During the same period, netrin-1 protein was detected along the trajectory of olfactory axons up to the olfactory bulb and its expression pattern in the nasal mesenchyme largely overlapped that of DCC. Moreover, netrin-1 continued to be present during the two first post-natal weeks, and a weak protein expression still persisted in the dorso-medial region of the olfactory epithelium in adult rats. While unilateral bulbectomy induced a transient up-regulation of netrin-1 in the lamina propria, particularly in the dorso-medial region of the neuroepithelium, no DCC expression was detected on the regenerating olfactory axons. In the developing olfactory bulb, the extension of mitral cell axons was associated with DCC presence while netrin-1 was absent along this axonal path. DCC was also highly expressed in the newly formed glomeruli after birth, and a weak DCC expression was still detected in the glomerular layer in adult rats. Taken together, these data support the notion that netrin-1, via DCC expressed on axons, may play a role in promoting outgrowth and/or guidance of pioneering olfactory axons toward the olfactory bulb primordium. Moreover, association of netrin-1 with mesenchymal DCC may provide a permissive environment to the growth of both pioneer and later-growing axons. The maintenance of netrin-1 expression in the nasal mesenchyme of adult rats as well as its regional up-regulation following unilateral bulbectomy infer that netrin-1, even in the absence of DCC, may be involved in the process of axonal growth of newly differentiated olfactory receptor neurons probably through the use of other receptors.

Down-regulation of T1A12/mac25, a novel insulin-like growth factor binding protein related gene, is associated with disease progression in breast carcinomas.

To define genes that are essential to the initiation and progression of breast cancer we utilized subtractive hybridization and differential display cloning techniques and isolated over 950 cDNAs from breast cell-lines derived from matched normal and tumor tissue. Of these, 102 cDNAs were characterized by DNA sequencing and Northern blot analysis. GenBank searches showed that one of these genes, T1A12 is identical to mac25, an insulin-like growth factor-binding protein related gene. Antibodies generated against the C-terminal region of the T1A12/mac25 protein were used to investigate its expression in 60 primary breast tissues. Sections of 12 benign, 16 ductal carcinoma in situ and 32 infiltrating ductal carcinoma specimens were examined. Strong immunoperoxidase staining was observed in luminal epithelial cells of normal lobules and ducts, in apocrine cells of cysts and fibroadenomas. Moderate to weak protein expression was found in hyperplastic and DCIS cells, but no specific staining was detected in invasive carcinoma cells. FISH mapping using a PAC clone localized the T1A12/mac25 gene to 4q12-13. Microsatellite length polymorphism was studied using markers for 4q in paired normal and tumor breast tissues. Thirty-three per cent (10/30) of the samples were found to be polymorphic with D4S189 and D4S231 microsatellite markers and LOH was detected in 50% (5/10) of these informative samples. Our data indicate that T1A12/mac25 expression is abrogated during breast cancer progression concomitant with loss of heterozygosity on chromosome 4q. T1A12/mac25 may therefore have a tumor suppressor-like function and its expression could indicate a disease with a more favorable status, having a better prognosis.

Structural and Functional Correlates between HIV-1 and SIV Nef Isolates

Thenefgenes of HIV-1 and SIV encode 27–34 kDa myristoylated proteins which have been shown to induce cell surface CD4 downregulation and bind to a cellular protein kinase. To identify regions of Nef important for function, structure–function correlates of HIVSF2nef(Nef) and SIVmm239opennef(SNef) were sought by constructing Nef/SNef hybrids. Metabolic labeling with35[S]methionine/cysteine demonstrated similar amounts of35[S] incorporation into all but one hybrid, SNeftll, in which the C-terminus of SNef was replaced by that of Nef. The weak protein expression of SNeftll was attributable to its short half-life of approximately 45 min. Nef, SNef, and SHSNef, a hybrid containing the internal sequences of Nef and the N- and C-terminal sequences from SNef, downregulated CD4 in human CEM cells. Only Nef and SHSNef downregulated CD4 in mouse AKR1-G1 cells. Nef, SNef, and SHSNef also effectively bound phosphoproteins of MW = 62,000 and 78,000 in CEM cells. Two additional hybrids, in which the Nef sequences of SHSNef were replaced with additional SNef sequences, were essentially ineffective in both assays. Thus, in two different assays of Nef function, swapping the SIV and HIV internalnefsequences were shown to be greatly deleterious to Nef function while SHSNef remained functional.