Quantitative PET with 15 O provides absolute values for cerebral blood flow (CBF), cerebral blood volume (CBV), cerebral metabolic rate of oxygen (CMRO 2 ), and oxygen extraction fraction (OEF), which are used for assessment of brain pathophysiology. Absolute quantification relies on physically accurate measurement, which, thus far, has been achieved by 2-dimensional PET (2D PET), the current gold standard for measurement of CBF and oxygen metabolism. We investigated whether quantitative 15 O study with 3-dimensional PET (3D PET) shows the same degree of accuracy as 2D PET. Methods: 2D PET and 3D PET measurements were obtained on the same day on 8 healthy men (age, 21-24 y). 2D PET was performed using a PET scanner with bismuth germanate (BGO) detectors and a 150-mm axial field of view (FOV). For 3D PET, a 3D-only tomograph with gadolinium oxyorthosilicate (GSO) detectors and a 156-mm axial FOV was used. A hybrid scatter-correction method based on acquisition in the dual-energy window (hybrid dual-energy window [HDE] method) was applied in the 3D PET study. Each PET study included 3 sequential PET scans for C 15 O, 15 O 2 , and H 2 15 O (3-step method). The inhaled (or injected) dose for 3D PET was approximately one fourth of that for 2D PET. Results: In the 2D PET study, average gray matter values (mean 6 SD) of CBF, CBV, CMRO 2 , and OEF were 53 6 12 (mL/100 mL/min), 3.6 6 0.3 (mL/100 mL), 3.5 6 0.5 (mL/100 mL/min), and 0.35 6 0.06, respectively. In the 3D PET study, scatter correction strongly affected the results. Without scatter correction, average values were 44 6 6 (mL/100 mL/min), 5.2 6 0.6 (mL/100 mL), 3.3 6 0.4 (mL/100 mL/min), and 0.39 6 0.05, respectively. With the exception of OEF, values differed between 2D PET and 3D PET. However, average gray matter values of scatter-corrected 3D PET were comparable to those of 2D PET: 55 6 11 (mL/100 mL/min), 3.7 6 0.5 (mL/100 mL), 3.8 6 0.7 (mL/100 mL/min), and 0.36 6 0.06, respectively. Even though the 2 PET scanners with different crystal materials, data acquisition systems, spatial resolution, and attenuation-correction methods were used, the agreement of the results between 2D PET and scatter-corrected 3D PET was excellent. Conclusion: Scatter coincidence is a problem in 3D PET for quantitative 15 O study. The combination of both the present PET/CT device and the HDE scatter correction permits quantitative 3D PET with the same degree of accuracy as 2D PET and with a lower radiation dose. The present scanner is also applicable to conventional steady-state 15 O gas inhalation if inhaled doses are adjusted appropriately.
Background: Studies on the diversity of carbohydrate-binding proteins (lectins) are important in glycobiology. Results: A lectin having a novel primary structure was isolated from a mussel and found to have a globotriose-dependent cytotoxicity on Burkitt lymphoma cells. Conclusion: A new primary structure quite distinct from known lectin is described. Significance: Discovery of similar lectin structures from vertebrates will lead to progress in medical sciences.
MytiLec; a novel lectin isolated from the Mediterranean mussel (Mytilus galloprovincialis); shows strong binding affinity to globotriose (Gb3: Galα1-4Galβ1-4Glc). MytiLec revealed β-trefoil folding as also found in the ricin B-subunit type (R-type) lectin family, although the amino acid sequences were quite different. Classification of R-type lectin family members therefore needs to be based on conformation as well as on primary structure. MytiLec specifically killed Burkitt's lymphoma Ramos cells, which express Gb3. Fluorescein-labeling assay revealed that MytiLec was incorporated inside the cells. MytiLec treatment of Ramos cells resulted in activation of both classical MAPK/ extracellular signal-regulated kinase and extracellular signal-regulated kinase (MEK-ERK) and stress-activated (p38 kinase and JNK) Mitogen-activated protein kinases (MAPK) pathways. In the cells, MytiLec treatment triggered expression of tumor necrosis factor (TNF)-α (a ligand of death receptor-dependent apoptosis) and activation of mitochondria-controlling caspase-9 (initiator caspase) and caspase-3 (activator caspase). Experiments using the specific MEK inhibitor U0126 showed that MytiLec-induced phosphorylation of the MEK-ERK pathway up-regulated expression of the cyclin-dependent kinase inhibitor p21, leading to cell cycle arrest and TNF-α production. Activation of caspase-3 by MytiLec appeared to be regulated by multiple different pathways. Our findings, taken together, indicate that the novel R-type lectin MytiLec initiates programmed cell death of Burkitt’s lymphoma cells through multiple pathways (MAPK cascade, death receptor signaling; caspase activation) based on interaction of the lectin with Gb3-containing glycosphingolipid-enriched microdomains on the cell surface.
Freshly ovulated eggs are each surrounded by a compact cumulus oophorus. The overall diameter of the normal egg (including the zona pellucida) is about 100 μm. Cumulus cells, particularly those near the egg, are arranged redially in a viscous noncellular matrix. The spermatozoon is about 250 μm in length. The head a large acrosome, changes in which can be readily examined with the light (phase‐ contrast) microsope. When exposed to physiological salt solutions, testicular spermatozoa either were motionless or flexed the posterior half of their tails slowly. Spermatozoa from the caput epididymis were highly motile, flexing the entire tail. A few of them moved progressively. Mature spermatozoa from the vas deferens were highly motile and moved either straightforward or in a circle. They vibrated their tails stiffly without flexing them. In normally mated females, fertilization began sometime between 2 and 3 h after ovulation and was completed within the next 4 to 5 h. Spermatozoa swimming in the ampullary fluid or within the cumulus oophorus about the time of fertilization flexed the anterior half (which roughly corresponds to the midpieac region) of their tails. This peculiar movement may be homologous to the so‐called “hyperactivation” of spermatozoa as reported in several other mammalian species. Actively motile spermatozoa within the cumulus or no the zona pellucida had either modified (“collapsed”) or no acrosomal caps. The sperm head usually passed verticually or nearly through the zona, but the path was oblique in some instances. In 54% of the recently fertilized eggs examined, the entire length of the sperm tail was within the perivitelline space; in the other 46% of the eggs varying lenghts of the tail remined the perivitelline space, the tails were extruded from the vitellus of many eggs even before the eggs began their first cleavage. When unfertilized eggs in the cumulus oophorus were inseminated with vas deferens spermatozoa in a modified Tyrode's solution (m‐TALP), about 80% of them were ferrtilized by 4–6 h after insemination. The vast majority were monospermic. When eggs were freed from the cumulus prior to insemination, none were fertilized, suggesting that the cumulus cells or their matrix assisted capacitation and/or the acrosome reaction of the spermatozoa under the in vitro conditions employed. No eggs were fertilized by the testicular or caput epididymal spermatozoa regardless of the presence or absence of cumulus oophorus around the eggs at the time of insemination.
Sialic acid binding lectin (SBL) isolated from Rana catesbeiana oocytes is a multifunctional protein which has lectin activity, ribonuclease activity and antitumor activity. However, the mechanism of antitumor effects of SBL is unclear to date and the validity for human leukemia cells has not been fully studied. We report here that SBL shows cytotoxicity for some human leukemia cell lines including multidrug-resistant (MDR) cells. The precise mechanisms of SBL-induced apoptotic signals were analyzed by combinational usage of specific caspase inhibitors and the mitochondrial membrane depolarization detector JC-1. It was demonstrated that SBL causes mitochondrial perturbation and the apoptotic signal is amplified by caspases and cell death is executed in a caspase-dependent manner. The efficacy of this combinational usage was shown for the first time, to distinguish the apoptotic pathway in detail. SBL selectively kills tumor cells, is able to exhibit cytotoxicity regardless of P-glycoprotein expression and has potential as an alternative to conventional DNA-damaging anticancer drugs.
Malignant mesothelioma is a highly aggressive tumor with poor prognosis. An effective drug for treatment of malignant mesothelioma is greatly needed. Sialic acid-binding lectin (SBL) isolated from oocytes of Rana catesbeiana is a multifunctional protein which has lectin activity, ribonuclease activity and antitumor activity, so it could be developed as a new type of anticancer drug. The validity of SBL for treatment of malignant mesothelioma was assessed using three malignant mesotheliomas and a non-malignant mesothlial cell line. Effectiveness of combinatorial treatment of SBL and tumor necrosis factor-related apoptosis inducing ligand (TRAIL) was also elucidated and characterized. SBL induced tumor-selective cytotoxicity that was attributed to induction of apoptosis. Combinatorial treatment of SBL and TRAIL showed synergistic apoptosis-inducing effect. Additional experiments revealed that Bid was the mediating molecule for the synergistic effect in SBL and TRAIL. These results suggested that SBL could be a promising candidate for the therapeutics for malignant mesothelioma. Furthermore, the combinatorial treatment of SBL and TRAIL could be an effective regimen against malignant mesothelioma.
A divalent cation-independent 16 kDa D-galactose binding lectin (AKL-2) was isolated from eggs of sea hare, Aplysia kurodai. The lectin recognized D-galactose and D-galacturonic acid and had a 32 kDa dimer consisting of two disulfide-bonded 16 kDa subunits. Eighteen N-terminus amino acids were identified by Edman degradation, having unique primary structure. Lectin blotting analysis with horseradish peroxidase-conjugated lectins has shown that AKL-2 was a glycoprotein with complex type oligosaccharides with N-acetyl D-glucosamine and mannose at non-reducing terminal. Two protein bands with 38 and 36 kDa in the crude extract of sea hare eggs after purification of the lectin was isolated by AKL-2-conjugated Sepharose column and elution with 0.1 M lactose containing buffer. It suggested that the lectin binds with an endogenous ligand in the eggs. AKL-2 kept extreme stability on haemagglutination activity if it was treated at pH 3 and 70 °C for 1 h. Glycan binding profile of AKL-2 by frontal affinity chromatography technology using 15 pyridylamine labeled oligosaccharides has been appeared that the lectin uniquely recognized globotriose (Galα1-4Galβ1-4Glc; Gb3) in addition to bi-antennary complex type N-linked oligosaccharides with N-acetyllactosamine. Surface plasmon resonance analysis of AKL-2 against a neo-glycoprotein, Gb3-human serum albumin showed the k(ass) and k(diss) values are 2.4 × 10³ M⁻¹ s⁻¹ and 3.8 × 10⁻³ s⁻¹, respectively. AKL-2 appeared cytotoxicity against both Burkitt's lymphoma Raji cell and erythroleukemia K562. The activity to Raji by the lectin was preferably cancelled by the co-presence of melibiose mimicing Gb3. On the other hand, K562 was cancelled effectively by lactose than melibiose. It elucidated that AKL-2 had cytotoxic ability mediated glycans structure to cultured cells.
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