Covalent attachment of poly(ethylene glycol) (PEG) molecules to drugs, proteins, and liposomes is a proven technology for improving their bioavailability, safety, and efficacy. Qualitative and quantitative analysis of PEG-derivatized molecules is important for both drug development and clinical applications. We previously reported the development of a monoclonal IgM antibody (AGP3) to PEG. We now describe a new IgG1 monoclonal antibody (E11) to PEG and show that it can be used in combination with AGP3 to detect and quantify PEG-derivatized molecules. Both antibodies bound the repeating subunits of the PEG backbone and could detect free PEG and PEG-modified proteins by ELISA, immunoblotting, and flow cytometry. Detection sensitivity increased with the length and the number of PEG chains on pegylated molecules. Both antibodies also efficiently accelerated the clearance of a PEG-modified enzyme in vivo. A sandwich ELISA in which E11/AGP3 were employed as the capture/detection antibodies was developed to detect PEG-modified proteins at concentrations as low as 1.2 ng/mL. In addition, the ELISA could also quantify, in the presence of 10% fetal bovine serum, free methoxy-PEG20,000, PEG2,000-quantum dots, and PEG2,000-liposomes at concentrations as low as 20 ng/mL (1.0 nM), 1.4 ng/mL (3.1 pM), and 2.4 ng/mL (3.13 nM phospholipids), respectively. Finally, we show that the sandwich ELISA could accurately measured the in vivo half-life of a PEG-modified enzyme. These antibodies should be generally applicable to the qualitative and quantitative analysis of all PEG-derivatized molecules.

No abstract

These findings also highlight the need to prompt further prospective studies on more cases of CRC to further establish the clinical relevance of activating KRAS mutation detection from peripheral blood in anti- EGFR-based chemotherapy that uses activating KRAS detection chips and the WCHMA analysis method.

The importance of nitric oxide (NO) in mediating vasodilation, neurotransmission, and immune and inflammatory responses has been demonstrated. Human keratinocyte express inducible nitric oxide synthase (iNOS) and the neuronal constitutive isoform of NOS (ncNOS). We established an in vitro model in keratinocytes to investigate changes in NO, iNOS and ncNOS expression after UVB exposure. We demonstrated a large induction of NO after UVB exposure and that the source of NO produced in UVB-exposed keratinocytes was increased expression of iNOS and ncNOS. The increased NO production with increased expression of iNOS and ncNOS may contribute to the pathological and physiological features of UVB-induced erythema and skin inflammation.

No abstract

No abstract

The HCMV IE2 protein negatively autoregulates its own expression as well as represses the transactivation activity of p53. Using the repression domain of IE2 as bait in the yeast two-hybrid system, Nrf1 and Nrf2, members of the CNC-bZIP family, were found to be IE2-interacting proteins. Residues 331-448 encompassing the DNA-binding and the dimerization domains of Nrf1 are sufficient for the interaction. The interaction was further confirmed in vitro by a glutathione S-transferase pull-down assay and in vivo by co-immunoprecipitation. In transient transfection studies, transcription driven by six copies of an NF-E2 site or by chimeric proteins between the DNA-binding domain of LexA and members of the CNC-bZIP family is repressed by IE2. Importantly, the DNA binding activity of the Nrf1/MafK heterodimer is not impeded by IE2. In a parallel study, CNC-bZIP factors attenuate the negative autoregulation of IE2. The attenuation could be explained by the finding that Nrf1 functions alone and synergistically with its heterodimerization partner, MafK, in inhibiting the DNA binding activity of IE2. Taken together, these results demonstrate the existence of antagonism between members of the CNC-bZIP family and IE2.Human cytomegalovirus (HCMV), 1 a member of the ␤ subgroup of herpesviruses, has a double-stranded DNA genome of 229,354 base pairs with a potential to encode more than 200 proteins (1). A number of immediate-early (IE) proteins of HCMV are made following entry of the virus into cells (2). Among them the IE2 86-kDa protein (referred hereafter as IE2) is most studied. IE2 appears to be a promiscuous transactivator of viral and cellular gene expression (3-5). IE2 stimulates transcription by interacting with general transcription factors and/or gene-specific factors (6 -9). In addition, IE2 negatively autoregulates its own expression by binding to a short nucleotide sequence, termed the cis repression signal (CRS), located immediately downstream of the TATA box of the HCMV major immediate early promoter (MIEP) (10, 11). Binding of IE2 to CRS prevents the recruitment of RNA polymerase II to the promoter via steric hindrance (12). Thus, IE2-mediated, CRSdependent transcriptional repression is both passive and position-dependent. The efficiency of IE2-mediated repression of MIEP seems to be cell-dependent (4, 10, 13), strongly suggesting that the IE2 activity may be modulated by post-translational modification and/or by interaction with cellular proteins. Indeed, phosphorylation has been shown to be one of these factors (14).Furthermore, recent studies demonstrate that IE2 can be converted into a trans-repressor following binding to p53 (15,16). A transcriptional repression domain has been mapped to the C terminus of IE2 (16), which is also required for its transcriptional activation, DNA binding, and interaction with CREB, c-Jun, RB, TBP, and TFIIB (6 -9). The DNA-binding and the repression domains of IE2 seem to be overlapping, because an IE2 mutant devoid of DNA binding activity also loses its repression activity (1...

Beta-adrenergic receptor-mediated relaxation of blood vessels declines in lead induced hypertension although the mechanism is unknown. We have utilized the aorta of lead hypertensive rats to investigate this problem. In an effort to elucidate the mechanism responsible for this alteration we examined plasma catecholamine levels, vascular beta-adrenergic receptor density, and cyclic adenosine monophosphate (cAMP) production in lead hypertensive rats. The density of beta-adrenergic receptors was 41% lower in the blood vessels of lead hypertensive rats compared with control rats. The corresponding apparent Kd values were not significantly different between two groups. The plasma catecholamine level was significantly higher in lead hypertensive rats compared with controls (P < 0.001). Stimulation of the vascular beta-adrenoceptor resulted in significantly lower levels of cAMP in lead hypertensive rats compared with controls (P < 0.001). The present results suggest that there is reduced beta-adrenoceptor density and diminished cAMP accumulation in blood vessels from lead hypertensive rats. Plasma catecholamine may play a role in the diminished beta-adrenoceptor and responsiveness to cAMP-mediated vascular relaxation in lead exposure.