Synthesis and two-photon photolysis of 6-(ortho-nitroveratryl)-caged IP3 in living cells

The synthesis of a photolabile derivative of inositol-1,4,5-trisphosphate (IP3) is described. This new caged second messenger (6-ortho-nitroveratryl)-IP3 (6-NV-IP3) has an extinction coefficient of 5000 M(-1) cm(-1) at 350 nm, and a quantum yield of photolysis of 0.12. Therefore, 6-NV-IP3 is photolyzed with UV light about three times more efficiently than the widely used P(4(5))-1-(2-nitrophenyl)ethyl-caged IP3 (NPE-IP3). 6-NV-IP3 has a two-photon cross-section of about 0.035 GM at 730 nm. This absorbance is sufficiently large for effective two-photon excitation in living cells at modest power levels. Using near-IR light (5 mW, 710 nm, 80 MHz, pulse-width 70 fs), we produced focal bursts of IP3 in HeLa cells, as revealed by laser-scanning confocal imaging of intracellular Ca2+ concentrations. Therefore, 6-NV-IP3 can be used for efficient, subcellular photorelease of IP3, not only in cultured cells but also, potentially, in vivo. It is in the latter situation that two-photon photolysis should reveal its true forte.

The nitrodibenzofuran chromophore: a new caging group for ultra-efficient photolysis in living cells

Photochemical uncaging of bio-active molecules was introduced in 1977, but since then, there has been no substantial improvement in the properties of generic caging chromophores. We have developed a new chromophore, nitrodibenzofuran (NDBF) for ultra-efficient uncaging of second messengers inside cells. Photolysis of a NDBF derivative of EGTA (caged calcium) is about 16-160 times more efficient than photolysis of the most widely used caged compounds (the quantum yield of photolysis is 0.7 and the extinction coefficient is 18,400 M(-1) cm(-1)). Ultraviolet (UV)-laser photolysis of NDBF-EGTA:Ca(2+) rapidly released Ca(2+) (rate of 20,000 s(-1)) and initiated contraction of skinned guinea pig cardiac muscle. NDBF-EGTA has a two-photon cross-section of approximately 0.6 GM and two-photon photolysis induced localized Ca(2+)-induced Ca(2+) release from the sarcoplasmic recticulum of intact cardiac myocytes. Thus, the NDBF chromophore has great promise as a generic and photochemically efficient protecting group for both one- and two-photon uncaging in living cells.

Hint2, a mitochondrial apoptotic sensitizer down-regulated in hepatocellular carcinoma

BACKGROUND ; AIMS: Hints, histidine triad nucleotide-binding proteins, are adenosine monophosphate-lysine hydrolases of uncertain biological function. Here we report the characterization of human Hint2. METHODS: Tissue distribution was determined by real-time quantitative polymerase chain reaction and immunoblotting, cellular localization by immunocytochemistry, and transfection with green fluorescent protein constructs. Enzymatic activities for protein kinase C and adenosine phosphoramidase in the presence of Hint2 were measured. HepG2 cell lines with Hint2 overexpressed or knocked down were established. Apoptosis was assessed by immunoblotting for caspases and by flow cytometry. Tumor growth was measured in SCID mice. Expression in human tumors was investigated by microarrays. RESULTS: Hint2 was predominantly expressed in liver and pancreas. Hint2 was localized in mitochondria. Hint2 hydrolyzed adenosine monophosphate linked to an amino group (AMP-pNA; k(cat):0.0223 s(-1); Km:128 micromol/L). Exposed to apoptotic stress, fewer HepG2 cells overexpressing Hint2 remained viable (32.2 +/- 0.6% vs 57.7 +/- 4.6%), and more cells displayed changes of the mitochondrial membrane potential (87.8 +/- 2.35 vs 49.7 +/- 1.6%) with more cleaved caspases than control cells. The opposite was observed in HepG2 cells with knockdown expression of Hint2. Subcutaneous injection of HepG2 cells overexpressing Hint2 in SCID mice resulted in smaller tumors (0.32 +/- 0.13 g vs 0.85 +/- 0.35 g). Microarray analyses revealed that HINT2 messenger RNA is downregulated in hepatocellular carcinomas (-0.42 +/- 0.58 log2 vs -0.11 +/- 0.28 log2). Low abundance of HINT2 messenger RNA was associated with poor survival. CONCLUSION: Hint2 defines a novel class of mitochondrial apoptotic sensitizers down-regulated in hepatocellular carcinoma.

Apparent diffusion coefficient in the aging mouse brain: a magnetic resonance imaging study

Novel magnetic resonance imaging sequences have and still continue to play an increasing role in neuroimaging and neuroscience. Among these techniques, diffusion-weighted imaging (DWI) has revolutionized the diagnosis and management of diseases such as stroke, neoplastic disease and inflammation. However, the effects of aging on diffusion are yet to be determined. To establish reference values for future experimental mouse studies we tested the hypothesis that absolute apparent diffusion coefficients (ADC) of the normal brain change with age. A total of 41 healthy mice were examined by T2-weighted imaging and DWI. For each animal ADC frequency histograms (i) of the whole brain were calculated on a voxel-by-voxel basis and region-of-interest (ROI) measurements (ii) performed and related to the animals' age. The mean entire brain ADC of mice <3 months was 0.715(+/-0.016) x 10(-3) mm2/s, no significant difference to mice aged 4 to 5 months (0.736(+/-0.040) x 10(-3) mm2/s) or animals older than 9 months 0.736(+/-0.020) x 10(-3) mm2/s. Mean whole brain ADCs showed a trend towards lower values with aging but both methods (i + ii) did not reveal a significant correlation with age. ROI measurements in predefined areas: 0.723(+/-0.057) x 10(-3) mm2/s in the parietal lobe, 0.659(+/-0.037) x 10(-3) mm2/s in the striatum and 0.679(+/-0.056) x 10(-3) mm2/s in the temporal lobe. With advancing age, we observed minimal diffusion changes in the whole mouse brain as well as in three ROIs by determination of ADCs. According to our data ADCs remain nearly constant during the aging process of the brain with a small but statistically non-significant trend towards a decreased diffusion in older animals.

Contribution of the apparent diffusion coefficient in perilesional edema for the assessment of brain tumors

OBJECTIVES: Diffusion-weighted MRI is sensitive to molecular motion and has been applied to the diagnosis of stroke. Our intention was to investigate its usefulness in patients with brain tumor and, in particular, in the perilesional edema. METHODS: We performed MRI of the brain, including diffusion-weighted imaging and mapping of the apparent diffusion coefficient (ADC), in 16 patients with brain tumors (glioblastomas, low-grade gliomas and metastases). ADC values were determined by the use of regions of interest positioned in areas of high signal intensities as seen on T2-weighted images and ADC maps. Measurements were taken in the tumor itself, in the area of perilesional edema and in the healthy contralateral brain. RESULTS: ADC mapping showed higher values of peritumoral edema in patients with glioblastoma (1.75 x 10(-3)mm(2)/s) and metastatic lesions (1.61 x 10(-3)mm(2)/s) compared with those who had low-grade glioma (1.40 x10(-3)mm(2)/s). The higher ADC values in the peritumoral zone were associated with lower ADC values in the tumor itself. CONCLUSIONS: The higher ADC values in the more malignant tumors probably reflect vasogenic edema, thereby allowing their differentiation from other lesions.

Diffusion-weighted imaging of the parotid gland: Influence of the choice of b-values on the apparent diffusion coefficient value

PURPOSE: To determine how the ADC value of parotid glands is influenced by the choice of b-values. MATERIALS AND METHODS: In eight healthy volunteers, diffusion-weighted echo-planar imaging (DW-EPI) was performed on a 1.5 T system, with b-values (in seconds/mm2) of 0, 50, 100, 150, 200, 250, 300, 500, 750, and 1000. ADC values were calculated by two alternative methods (exponential vs. logarithmic fit) from five different sets of b-values: (A) all b-values; (B) b=0, 50, and 100; (C) b=0 and 750; (D) b=0, 500, and 1000; and (E) b=500, 750, and 1000. RESULTS: The mean ADC values for the different settings were (in 10(-3) mm2/second, exponential fit): (A) 0.732+/-0.019, (B) 2.074+/-0.084, (C) 0.947+/-0.020, (D) 0.890+/-0.023, and (E) 0.581+/-0.021. ADC values were significantly (P <0.001) different for all pairwise comparisons of settings (A-E) of b-values, except for A vs. D (P=0.172) and C vs. D (P=0.380). The ADC(B) was significantly higher than ADC(C) or ADC(D), which was significantly higher than ADC(E). ADC values from exponential vs. logarithmic fit (P=0.542), as well as left vs. right parotid gland (P=0.962), were indistinguishable. CONCLUSION: The ADC values calculated from low b-value settings were significantly higher than those calculated from high b-value settings. These results suggest that not only true diffusion but also perfusion and saliva flow may contribute to the ADC.