Generation of GFP Native Protein for Detection of Its Intracellular Uptake by Cell-Penetrating Peptides.

Different types of lipid- and polymer-based vectors have been developed to deliver proteins into cells, but these methods showed relatively poor efficiency. Recently, a group of short, highly basic peptides known as cell-penetrating peptides (CPPs) were used to carry polypeptides and proteins into cells. In this study, expression and purification of GFP protein was performed using the prokaryotic pET expression system. We used two amphipathic CPPs (Pep-1 and CADY-2) as a novel delivery system to transfer the GFP protein into cells. The morphological features of the CPP/GFP complexes were studied by scanning electron microscopy (SEM), Zetasizer, and SDS-PAGE. The efficiency of GFP transfection using Pep-1 and CADY-2 peptides and TurboFect reagent was compared with FITC-antibody protein control delivered by these transfection vehicles in the HEK-293T cell line. SEM data confirmed formation of discrete nanoparticles with a diameter of below 300 nm. Moreover, formation of the complexes was detected using SDS-PAGE as two individual bands, indicating non-covalent interaction. The size and homogeneity of Pep-1/GFP and CADY-2/GFP complexes were dependent on the ratio of peptide/cargo formulations, and responsible for their biological efficiency. The cells transfected by Pep-1/GFP and CADY-2/GFP complexes at a molar ratio of 20 : 1 demonstrated spreading green regions using fluorescent microscopy. Flow cytometry results showed that the transfection efficiency of Pep-based nanoparticles was similar to CADY-based nanoparticles and comparable with TurboFect-protein complexes. These data open an efficient way for future therapeutic purposes.

The impact of p16 and HER2 expression on survival in patients with ovarian carcinoma.

The purpose of this study was to investigate the impact of p16 and HER2 expression on survival in patients with ovarian carcinoma.This descriptive-analytic, cross-sectional study, was conducted on 47 paraffin blocks of epithelial ovarian tumors. Suitable slides were prepared to evaluate HER2 and p16 by immunohistochemistry using HercepTest kit (DAKO) and p16INK4A kit (DAKO, code 5334). Clinical information and pathology data were extracted from patients' medical and pathology records. Data entry and analysis was done by SPSS (version 16) software. Chi-square test, Mann-Whitney test, t-test, Kruskal-Wallis test, log rank test and Kaplan-Meier method were used. The mean age of the patients was 51.6 years (range 19-71 years). The most common histological type of epithelial ovarian cancer was serous adenocarcinoma (68.1%). P16 expression was detected in 34% of epithelial ovarian tumors. P16 expression was significantly associated with stage of disease (P = 0.04) and overall survival (P = 0.001), but HER2 expression was not associated with overall survival, stage of disease and tumor histological type.Expression of p16 may be used as a prognostic factor of overall survival and stage of disease, while HER2 expression may not be used as a prognostic factor of overall survival.

Epidemiologic and clinicopathologic evaluation of patients with breast cancer referred to Ghaem Hospital from 2005 to 2014.

Introduction: The most common cancer is Breast Cancer and the first principal purpose of cancer deaths in females of 44-40 ages. The currency of three negative breast cancer involves 10-17%. his sort cancer of the breast is described by a negative receptor of estrogen, progesterone, and HER2 that is much more competitive than the other kinds and the forecast is weaker. Therefore, this research was conducted with the aim of evaluating the outcomes of the medication in patients with a triple negative sort of breast cancer about the different patients with breast cancer. Method: This historical group research was conducted by leading to the reports of all patients with breast cancer whose medication and follow-up was conducted in Hospital of Mashhad Ghaem through 2001 and 2010, their ER, PR, HER2 outcomes being reported in the files. Based on immunohistochemical records (ER, PR, HER2), patients were split into 2 collections: triple negative and another negative and the therapeutic results were analyzed among the 2 groups regarding two and five-year disease-free and throughout durability by Kaplan-Meier method. P<0.05 was regarded necessary. Results: The medicinal studies of 331 cases with breast cancer were investigated in this research. The number of members in the Triple negative collection was 101 (30.5%) and in another negative group was 230 (69.5%). The base overall durability in the triple negative was 32.48 ± 24.56 months and in another negative was 29.67 ± 22.36 months and no notable variation was recognized in the studied collections (P=0.306). Furthermore, the low disease-free durability in the triple negative group was 30.57 ± 24.56 months and in another negative was 28.21 ± 21.72 months and no important variation was recognized in the studied collections (P=0.184). Conclusion: under previously directed investigations, between all the samples of breast cancer, Triple negative had a weaker forecast and a lower durability with the cases in our research, the overall durability and disease-free longevity got were the same in 2 collections of Triple negative and another negative, and the basis of this relationship was apparently the residence of HER2 + subgroup in the non-Triple negative collection which pointed to the durability of cases in the non-Triple negative collection be similar to the Triple negative collection.

Sentinel node biopsy for lymph nodal staging of uterine cervix cancer: a systematic review and meta-analysis of the pertinent literature.

BACKGROUND: We reviewed the available literature on the accuracy of sentinel node mapping in the lymph nodal staging of uterine cervical cancers. METHODS: MEDLINE and Scopus were searched by using "sentinel AND (cervix OR cervical)" as key words. Studies evaluating the accuracy of sentinel node mapping in the lymph nodal staging of uterine cervical cancers were included if enough data could be extracted for calculation of detection rate and/or sensitivity. RESULTS: Sixty-seven studies were included in the systematic review. Pooled detection rate was 89.2% [95% CI: 86.3-91.6]. Pooled sensitivity was 90% [95% CI: 88-92]. Sentinel node detection rate and sensitivity were related to mapping method (blue dye, radiotracer, or both) and history of pre-operative neoadjuvant chemotherapy. Sensitivity was higher in patients with bilaterally detected pelvic sentinel nodes compared to those with unilateral sentinel nodes. Lymphatic mapping could identify sentinel nodes outside the routine lymphadenectomy limits. CONCLUSION: Sentinel node mapping is an accurate method for the assessment of lymph nodal involvement in uterine cervical cancers. Selection of a population with small tumor size and lower stage will ensure the lowest false negative rate. Lymphatic mapping can also detect sentinel nodes outside of routine lymphadenectomy areas providing additional histological information which can improve the staging. Further studies are needed to explore the impact of sentinel node mapping in fertility sparing surgery and in patients with history of neoadjuvant chemotherapy.

Restoration of nucleotide excision repair in a helicase-deficient XPD mutant from intragenic suppression by a trichothiodystrophy mutation.

The UV-sensitive V-H1 cell line has a T46I substitution mutation in the Walker A box in both alleles of XPD and lacks DNA helicase activity. We characterized three partial revertants that curiously display intermediate UV cytotoxicity (2- to 2.5-fold) but normal levels of UV-induced hprt mutations. In revertant RH1-26, the efficient removal of pyrimidine (6-4) pyrimidone photoproducts from both strands of hprt suggests that global-genomic nucleotide excision repair is normal, but the pattern of cyclobutane pyrimidine dimer removal suggests that transcription-coupled repair (TCR) is impaired. To explain the intermediate UV survival and lack of RNA synthesis recovery in RH1-26 after 10 J of UV/m(2), we propose a defect in repair-transcription coupling, i.e., the inability of the cells to resume or reinitiate transcription after the first TCR event within a transcript. All three revertants carry an R658H suppressor mutation, in one allele of revertants RH1-26 and RH1-53 and in both alleles of revertant RH1-3. Remarkably, the R658H mutation produces the clinical phenotype of trichothiodystrophy (TTD) in several patients who display intermediate UV sensitivity. The XPD(R658H) TTD protein, like XPD(T46I/R658H), is codominant when overexpressed in V-H1 cells and partially complements their UV sensitivity. Thus, the suppressing R658H substitution must restore helicase activity to the inactive XPD(T46I) protein. Based on current knowledge of helicase structure, the intragenic reversion mutation may partially compensate for the T46I mutation by perturbing the XPD structure in a way that counteracts the effect of this mutation. These findings have implications for understanding the differences between xeroderma pigmentosum and TTD and illustrate the value of suppressor genetics for studying helicase structure-function relationships.

Codominance associated with overexpression of certain XPD mutations.

Mutations in the XPD gene are associated with three complex clinical phenotypes, namely xeroderma pigmentosum (XP), XP in combination with Cockayne syndrome (XP-CS), and trichothiodystrophy (TTD). XP is caused by a deficiency in nucleotide excision repair (NER) that results in a high risk of skin cancer. TTD is characterized by severe developmental and neurological defects, with hallmark features of brittle hair and scaly skin, and sometimes has defective NER. We used CHO cells as a system to study how specific mutations alter the dominant/recessive behavior of XPD protein. Previously we identified the T46I and R75W mutations in two highly UV-sensitive hamster cell lines that were reported to have paradoxically high levels of unscheduled DNA synthesis. Here we report that these mutants have greatly reduced XPD helicase activity and fully defective NER in a cell-extract excision assay. We conclude that the unscheduled DNA synthesis seen in these mutants is caused by abortive "repair" that does not contribute to cell survival. These mutations, as well as the K48R canonical helicase-domain mutation, each produced codominant negative phenotypes when overexpressed in wild-type CHO cells. The common XP-specific R683W mutation also behaved in a codominant manner when overexpressed, which is consistent with the idea that this mutation may affect primarily the enzymatic activity of the protein rather than impairing protein interactions, which may underlie TTD. A C-terminal mutation uniquely found in TTD (R722W) was overexpressed but not to levels sufficiently high to rigorously test for a codominant phenotype. Overexpression of mutant XPD alleles may provide a simple means of producing NER deficiency in other cell lines.

Cloning, expression, and one-step purification of the minimal essential domain of the light chain of botulinum neurotoxin type A.

A truncated but functional form of the botulinum neurotoxin A light chain (Tyr 9-Leu 415) has been cloned into the three bacterial expression vectors, pET 28, pET 30, and PGEX-2T, and produced as fusion proteins. This 406-amino-acid light chain was expressed with 1 six-histidine tag (LC-pET28), 2 six histidine tags and a S-tag (LC-pET30), or a six-histidine tag and a glutathione S-transferase tag (LC-pGEX-2T). The three fusion proteins have been overexpressed in Escherichia coli, purified in a soluble form, and tested for protease activity. All three recombinant proteins were found to have similar enzymatic activity, comparable to the light chain purified from the whole toxin. The LC-pET30 protein was the most soluble and stable of the three fusion proteins, and it could be purified using a one-step affinity chromatography protocol. The purified protein was determined to be 98% pure as assessed by SDS-polyacrylamide gel. This protein has been crystallized and initial X-ray data show that the crystals diffract to 1.8 A.

Genetically modified Chinese hamster ovary cells for investigating sulfotransferase-mediated cytotoxicity and mutation by 2-amino-1-methyl-6- phenylimidazo[4,5-b]pyridine.

To test the hypothesis that the sulfotransferase gene plays a role in the phase II bioactivation of PhIP, a heterocyclic amine found in cooked meats, we transfected the UV5P3 cell line with cDNA plasmids of human aryl sulfotransferases (HAST1 and HAST3). UV5P3 is a nucleotide excision repair-deficient and P4501A2-expressing CHO cell line that we have previously developed. Functionally transformed clones were identified by the differential cytotoxicity (DC) assay that used PhIP as the cytotoxic agent. Two clones designated 5P3H1 and 5P3H3, expressing HAST1 and HAST3, respectively, were chosen for further characterization. Correct fragment sizes of the sulfotransferase cDNAs were identified in both cell lines by polymerase chain reaction. Immunoblot analysis confirmed the expression of the sulfotransferase proteins. The addition of the sulfotransferase inhibitor DCNP decreased the cytotoxic effects of PhIP in a dose-dependent manner. The increase in cell growth was 6. 5-fold for 5P3H1 and 2.4-fold for 5P3H3, relative to values obtained without DCNP. Based on D(50) values, the dose that reduced the survival to 50% relative to untreated controls, the cytotoxic effect of PhIP was increased threefold for 5P3H1 and 1.87-fold for 5P3H3 cell lines over the parental UV5P3 line. There was also a small increase in the mutation response at the aprt locus. These newly established 5P3H1 and 5P3H3 sulfotransferase-expressing cells provide valuable mechanistic information of the bioactivation of PhIP and related compounds. Environ. Mol. Mutagen. 35:57-65, 2000. Published 2000 Wiley-Liss, Inc.

Molecular analysis of ERCC2 mutations in the repair deficient hamster mutants UVL-1 and V-H1.

The cDNA sequence of the Chinese hamster ERCC2 nucleotide excision repair and transcription gene from the UVL-1 Chinese hamster ovary (CHO) mutant cell line and the V-H1 Chinese hamster V79 mutant line was analyzed. ERCC2 encodes a presumed ATP-dependent DNA helicase and is single copy in CHO lines due to the structural hemizygosity of chromosome 9. Both UVL-1 and V-H1 have intermediate levels of (6-4) photoproduct repair but are as highly UV sensitive as the group 2 mutants that have no detectable repair. Deficiency in cyclobutane dimer removal has also been shown for V-H1. In UVL-1, a single base substitution resulting in an Arg75-->Trp substitution in helicase domain Ia was identified. The equivalent amino acid position is also Arg in the human, mouse, Xiphophorus maculatus, Saccharomyces cerevisiae, and Schizosaccharomyces pombe homologs. In V-H1, a single base substitution resulting in a Thr46-->Ile substitution in helicase domain I (the ATP-binding domain) was identified in both alleles. The equivalent amino acid position is also Thr in the five homologs. Analysis of three V-H1 partial revertants revealed that they still have the original V-H1 mutation in both alleles, indicating that these are second site reversion events. Site-specific mutagenesis was used to introduce the Thr46-->Ile, Arg75-->Trp, and Lys48-->Arg (helicase domain I) mutations into a hamster ERCC2 expression plasmid. These plasmids each failed to confer UV resistance to group 2 mutant cells, further demonstrating that the changes identified are the causative mutations in V-H1 and UVL-1. Correlations between specific mutations, biochemical activities, and repair phenotype are discussed.

Construction of a functional cDNA clone of the hamster ERCC2 DNA repair and transcription gene.

The complete hamster ERCC2 cDNA was constructed in a plasmid vector from clones of three overlapping reverse transcribed/polymerase chain reaction amplified fragments using unique restriction enzyme recognition sites within the regions of overlap. This complete cDNA insert was then cloned into a mammalian expression vector, pcD2E, and tested for function by the ability to confer UV resistance to the ERCC2 mutant CHO cell line UV5. Site-specific mutagenesis was used to introduce the G347-->A and G1844-->A changes resulting in the Cys116-->Tyr and Gly615-->Glu mutations previously identified in UV5 and UVL-13 (also an ERCC2 mutant CHO cell line), respectively. The 116Tyr and 615Glu plasmids each failed to confer UV resistance to UV5 or UVL-13 cells, respectively, demonstrating that the changes identified are indeed the causative mutations in UV5 and UVL-13.

Uncoupling oxygen transfer and electron transfer in the oxygenation of camphor analogues by cytochrome P450-CAM. Direct observation of an intermolecular isotope effect for substrate C-H activation.

The hydroxylation of (1R)-camphor by cytochrome P450-CAM involves almost complete coupling of electron to oxygen transfer. Modifications at C-5 of camphor, the normal site of hydroxylation by P450-CAM, lead to as much as 98% uncoupling of electron and oxygen transfer as well as to decreases in the rate of electron uptake (up to 10-fold) and the rate of oxygenated product formation (up to 210-fold). Two modes of uncoupling are seen: (a) two-electron uncoupling in which the decrease in oxygenated product formation is balanced by increases in H2O2 formation and (b) four-electron "oxidase" uncoupling where the NADH/O2 ratio has changed from one to nearly two and relatively little H2O2 is formed. Both enantiomers of 5-methylenylcamphor are two-electron uncouplers, while (1R)- and (1S)-5,5-difluorocamphor and (1R)-9,9,9-d3-5,5-difluorocamphor are four-electron uncouplers. An intermolecular isotope effect of 11.7 is observed for oxygenation of C-9 in (1R)-5,5-difluorocamphor. With this substrate, the significant decrease in the rate of oxygenated product formation combined with the large isotope effect suggest that the rate-limiting step has switched from electron to oxygen transfer.

X-ray absorption near edge studies of cytochrome P-450-CAM, chloroperoxidase, and myoglobin. Direct evidence for the electron releasing character of a cysteine thiolate proximal ligand.

The low spin ferric and low and high spin ferrous forms of myoglobin, bacterial cytochrome P-450-CAM, and chloroperoxidase have been examined by Fe-K x-ray absorption edge spectroscopy. The positions of the absorption edge and the shapes of preedge and edge regions of imidazole adducts of ferric P-450-CAM and chloroperoxidase are essentially the same when compared with thiolate-ligated ferric myoglobin. As these three protein derivatives all have six-coordinate, low spin, ferric hemes with axial imidazole and thiolate ligands, the superposition of x-ray absorption edge spectral properties demonstrates that the protein environment does not effect the spectra, provided one compares heme iron centers with identical coordination numbers, spin and oxidation states, and ligand sets. In contrast, a 0.96 eV difference is observed in the energy of the absorption edge for imidazole- and thiolate-ligated ferric myoglobin with the latter shifted to lower energy as observed for ferrous myoglobin states. Similarly, in the low spin ferric-imidazole and ferrous-CO states, the energies of the absorption edge for chloroperoxidase and P-450-CAM are shifted in the direction of the ferrous state (to lower energy) when compared with those for analogous myoglobin derivatives. In the deoxyferrous high spin state, comparison of the edge spectra of chloroperoxidase with analogous data for cytochrome P-450-CAM suggests that the electron density at the iron is similar for these two protein states. The shifts observed in the energies of the x-ray absorption edge for the thiolate-ligated states of these proteins relative to derivatives lacking a thiolate ligand provide a direct measure of the electron releasing character of a thiolate axial ligand. These results therefore support the suggested role of the cysteinate proximal ligand of P-450 as a strong internal electron donor to promote O-O bond cleavage in the putative ferric-peroxide intermediate to generate the proposed ferryl-oxo "active oxygen" state of the reaction cycle.

Developmental regulation of flavin-containing monooxygenase (FMO) isoforms 1 and 2 in pregnant rabbit.

Mammalian flavin-containing monooxygenase (FMO, EC 1.14.13.8) metabolizes a vast number of structurally diverse xenobiotics containing a soft-nucleophile, typically a nitrogen or sulfur. FMO is not inducible by the classical cytochrome P450 (CYP) inducers, such as phenobarbital, polycyclic aromatic hydrocarbons, ethanol or macrolide antibiotics. Evidence does exist from a number of laboratories, however, for developmental and hormonal regulation of FMO. Our laboratory has confirmed previous observations of enhanced FMO activity during mid- and late-gestation in maternal rabbit lung and have demonstrated that this response is due to increased protein and catalytic activity associated with FMO2. The time course of expression of FMO2 during mid- and late-gestation correlates to plasma peaks of progesterone or cortisol. FMO2 also peaks at parturition in maternal kidney, coincident with plasma cortisol levels. FMO2 is induced by s.c. administration of either progesterone or dexamethasone in lung, or by dexamethasone in kidney. Correlation of plasma progesterone or cortisol levels during gestation and postpartum support a role for progesterone, but not cortisol in regulation of FMO2 in maternal rabbit lung. The levels of FMO1 also appear to be increased during mid- and late-gestation in liver. FMO1 in liver may also be regulated during gestation by progesterone or glucocorticoids as administration of these steroids enhanced FMO1 mRNA levels 4-fold.

On the use of iron octa-alkylporphyrins as models for protoporphyrin IX-containing heme systems in studies employing magnetic circular dichroism spectroscopy.

The magnetic circular dichroism (MCD) properties of numerous oxidation and ligation state derivatives of myoglobin and horseradish peroxidase reconstituted with an iron octa-alkylporphyrin (mesoheme IX) have been investigated in order to establish the utility of such porphyrins as models for protoporphyrin IX-containing systems. The MCD spectra of the mesoheme-reconstituted proteins are blue-shifted (4-12 nm) and are somewhat more intense (1.5-2.5 fold) when compared to the spectra of analogous derivatives of native myoglobin and horseradish peroxidase. However, the spectral band patterns of the mesoheme-reconstituted proteins closely resemble those of the native proteins in essentially all cases. These data demonstrate that octa-alkylporphyrins can be productively used as models for protoporphyrin IX in studies of heme proteins with MCD spectroscopy.

The active site structure of E. coli HPII catalase. Evidence favoring coordination of a tyrosinate proximal ligand to the chlorin iron.

E. coli produces 2 catalases known as HPI and HPII. While the heme prosthetic group of the HPII catalase has been established to be a dihydroporphyrin or chlorin, the identity of the proximal ligand to the iron has not been addressed. The magnetic circular dichroism (MCD) spectrum of native ferric HPII catalase is very similar to those of a 5-coordinate phenolate-ligated ferric chlorin complex, a model for tyrosinate proximal ligation, as well as of chlorin-reconstituted ferric horseradish peroxidase, a model for 5-coordinate histidine ligation. However, further MCD comparisons of chlorin-reconstituted myoglobin with parallel ligand-bound adducts of the catalase clearly rule out histidine ligation in the latter, leaving tyrosinate as the best candidate for the proximal ligand.

Cathepsin L--a latent proteinase in guinea pig sperm.

Guinea pig spermatozoa were found to contain a fully-latent cysteine proteinase that could be unmasked by incubating epididymal sperm for 2 hr at pH 3.5 and 37 degrees C. The proteinase was identified as cathepsin L (EC 3.4.22.15) on the basis of its optimal hydrolysis of benzyloxycarbonyl-Phe-Arg-7-(4-methyl)coumarylamide (Z-Phe-Arg-NMec) at pH 5.5; lack of action on Z-Arg-Arg-NMec and Arg-NMec; urea-enhanced digestion of azocasein; marked sensitivity to thiol reagents, leupeptin, Z-Phe-Phe-CHN2, and L-trans-epoxy-succinylleucylamido(3-methyl)butane (Ep-475 or E-64-c); and insensitivity to pepstatin and serine proteinase inhibitors. Gossypol, a male antifertility agent, was inhibitory. The unmasking phenomenon was reversibly inhibited by HgCl2 and mersalyl acid, and prevented by leupeptin and Ep-475, but not by pepstatin.