In vitro and in vivo evaluation of Alexa Fluor 680-bombesin[7-14]NH2 peptide conjugate, a high-affinity fluorescent probe with high selectivity for the gastrin-releasing peptide receptor

Gastrin-releasing peptide (GRP) receptors are overexpressed on several types of human cancer cells, including breast, prostate, small cell lung, and pancreatic cancers. Bombesin (BBN), a 14-amino acid peptide that is an analogue of human GRP, binds to GRP receptors with very high affinity and specificity. The aim of this study was to develop a new fluorescent probe based on BBN having high tumor uptake and optimal pharmacokinetics for specific targeting and optical imaging of human breast cancer tissue. In this study, solid-phase peptide synthesis was used to produce H(2)N-glycylglycylglycine-BBN[7-14]NH(2) peptide with the following general sequence: H(2)N-G-G-G-Q-W-A-V-G-H-L-M-(NH(2)). This conjugate was purified by reversed-phase high-performance liquid chromatography and characterized by electrospray-ionization mass spectra. The fluorescent probe Alexa Fluor 680-G-G-G-BBN[7-14]NH(2) conjugate was prepared by reaction of Alexa Fluor 680 succinimidyl ester to H(2)N-G-G-G-BBN[7-14]NH(2) in dimethylformamide (DMF). In vitro competitive binding assays, using (125)I-Tyr(4)-BBN as the radiolabeling gold standard, demonstrated an inhibitory concentration 50% value of 7.7 +/- 1.4 nM in human T-47D breast cancer cells. Confocal fluorescence microscopy images of Alexa Fluor 680-G-G-G-BBN[7-14]NH(2) in human T-47D breast cancer cells indicated specific uptake, internalization, and receptor blocking of the fluorescent bioprobe in vitro. In vivo investigations in SCID mice bearing xenografted T-47D breast cancer lesions demonstrated the ability of this new conjugate to specifically target tumor tissue with high selectivity and affinity.     

Accessibility of the distal heme face, rather than Fe-His bond strength, determines the heme-nitrosyl coordination number of cytochromes c': evidence from spectroscopic studies

The heme coordination chemistry and spectroscopic properties of Rhodobacter capsulatus cytochrome c' (RCCP) have been compared to data from Alcaligenes xylosoxidans (AXCP), with the aim of understanding the basis for their different reactivities with nitric oxide (NO). Whereas ferrous AXCP reacts with NO to form a predominantly five-coordinate heme-nitrosyl complex via a six-coordinate intermediate, RCCP forms an equilibrium mixture of six-coordinate and five-coordinate heme-nitrosyl species in approximately equal proportions. Ferrous RCCP and AXCP both exhibit high Fe-His stretching frequencies (227 and 231 cm(-)(1), respectively), suggesting that factors other than the Fe-His bond strength account for their differences in heme-nitrosyl coordination number. Resonance Raman spectra of ferrous-nitrosyl RCCP confirm the presence of both five-coordinate and six-coordinate heme-NO complexes. The six-coordinate heme-nitrosyl of RCCP exhibits a fairly typical Fe-NO stretching frequency (569 cm(-)(1)), in contrast to the relatively high value (579 cm(-)(1)) of the AXCP six-coordinate heme-nitrosyl intermediate. It is proposed that NO experiences greater steric hindrance in binding to the distal face of AXCP, as compared to RCCP, leading to a more distorted Fe-N-O geometry and an elevated Fe-NO stretching frequency. Evidence that RCCP has a more accessible distal coordination site than in AXCP stems from the fact that ferric RCCP readily forms a heme complex with exogenous imidazole, whereas AXCP does not. A model is proposed in which distal heme-face accessibility, rather than the proximal Fe-His bond strength, determines the heme-nitrosyl coordination number in cytochromes c'.     

Dietary soy and tea mitigate chronic inflammation and prostate cancer via NFκB pathway in the Noble rat model

Chronic inflammation and nuclear factor-kappa B (NFκB) have been implicated in prostate cancer development; thus, dietary factors that inhibit NFκB may serve as effective chemo-preventative agents. Prostate cancer risk is significantly lower in Asian countries compared to the United States, which has prompted interest in the potential chemopreventative action of Asian dietary components such as soy and green tea. This study examined the effects of dietary soy and tea on NFκB activation and inflammation in vivo using a hormone-induced rat model for prostate cancer. Male Noble rats implanted with estradiol and testosterone were divided into 4 dietary groups: control, soy, tea, or soy+tea. NFκB activation and inflammatory cytokines were measured post implantation. The combination of soy and tea suppressed NFκB p50 binding activity and protein levels via induction of IκBα. Soy and tea also decreased prostate inflammatory infiltration, increased Bax/BcL2 ratio and decreased protein expression of tumor necrosis factor-alpha, interleukin (IL)-6 and IL-1β compared to control. Soy and tea attenuated prostate malignancy by decreasing prostate hyperplasia. These effects were not apparent in groups treated with soy or tea alone. The ongoing in vivo studies thus far suggest that combination of foods, such as soy and tea, may inhibit hormone-induced proinflammatory NFκB signals that contribute to prostate cancer development.     

The use of unfixed cryostat sections for electron microscopic study of D-amino acid oxidase activity in rat liver.

Unfixed cryostat sections of rat liver were incubated to demonstrate D-amino acid oxidase activity at the ultrastructural level. Incubation was performed by mounting the sections on a semipermeable membrane which was stretched over a gelled incubation medium containing D-proline as substrate and cerium ions as capture reagent for hydrogen peroxide. After an incubation period of 30 min, ultrastructural morphology was retained to such an extent that the final reaction product could be localized in peroxisomes, whereas the crystalline core remained unstained. Control incubations were performed in the absence of substrate; the lack of final reaction product in peroxisomes indicated the specificity of the reaction. We conclude that the semipermeable membrane technique opens new perspectives for localization of enzyme activities at the ultrastructural level without prior tissue fixation, thus enabling localization of the activity of soluble and/or labile enzymes.     

Tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone promotes functional cooperation of Bcl2 and c-Myc through phosphorylation in regulating cell survival and proliferation

Nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is formed by nitrosation of nicotine and has been identified as the most potent carcinogen contained in cigarette smoke. NNK significantly contributes to smoking-related lung cancer, but the molecular mechanism remains enigmatic. Bcl2 and c-Myc are two major oncogenic proteins that cooperatively promote tumor development. We report here that NNK simultaneously stimulates Bcl2 phosphorylation exclusively at Ser(70) and c-Myc at Thr(58) and Ser(62) through activation of both ERK1/2 and PKCalpha, which is required for NNK-induced survival and proliferation of human lung cancer cells. Treatment of cells with staurosporine or PD98059 blocks both Bcl2 and c-Myc phosphorylation and results in suppression of NNK-induced proliferation. Specific depletion of c-Myc expression by RNA interference retards G(1)/S cell cycle transition and blocks NNK-induced cell proliferation. Phosphorylation of Bcl2 at Ser(70) promotes a direct interaction between Bcl2 and c-Myc in the nucleus and on the outer mitochondrial membrane that significantly enhances the half-life of the c-Myc protein. Thus, NNK-induced functional cooperation of Bcl2 and c-Myc in promoting cell survival and proliferation may occur in a novel mechanism involving their phosphorylation, which may lead to development of human lung cancer and/or chemoresistance.     

The occurrence of C(2) photosynthesis in Euphorbia subgenus Chamaesyce (Euphorbiaceae)

This study investigated whether Euphorbia subgenus Chamaesyce subsection Acutae contains C(3)-C(4) intermediate species utilizing C(2) photosynthesis, the process where photorespired CO(2) is concentrated into bundle sheath cells. Euphorbia species in subgenus Chamaesyce are generally C(4), but three species in subsection Acutae (E. acuta, E. angusta, and E. johnstonii) have C(3) isotopic ratios. Phylogenetically, subsection Acutae branches between basal C(3) clades within Euphorbia and the C(4) clade in subgenus Chamaesyce. Euphorbia angusta is C(3), as indicated by a photosynthetic CO(2) compensation point (Г) of 69 μmol mol(-1) at 30 °C, a lack of Kranz anatomy, and the occurrence of glycine decarboxylase in mesophyll tissues. Euphorbia acuta utilizes C(2) photosynthesis, as indicated by a Г of 33 μmol mol(-1) at 30 °C, Kranz-like anatomy with mitochondria restricted to the centripetal (inner) wall of the bundle sheath cells, and localization of glycine decarboxlyase to bundle sheath mitochondria. Low activities of PEP carboxylase, NADP malic enzyme, and NAD malic enzyme demonstrated no C(4) cycle activity occurs in E. acuta thereby classifying it as a Type I C(3)-C(4) intermediate. Kranz-like anatomy in E. johnstonii indicates it also utilizes C(2) photosynthesis. Given the phylogenetically intermediate position of E. acuta and E. johnstonii, these results support the hypothesis that C(2) photosynthesis is an evolutionary intermediate condition between C(3) and C(4) photosynthesis.