PMab-219: A monoclonal antibody for the immunohistochemical analysis of horse podoplanin

Monoclonal antibodies (mAbs) against human, mouse, rat, rabbit, dog, cat, and bovine podoplanin (PDPN), a lymphatic endothelial cell marker, have been established in our previous studies. However, mAbs against horse PDPN (horPDPN), which are useful for immunohistochemical analysis, remain to be developed. In the present study, mice were immunized with horPDPN-overexpressing Chinese hamster ovary (CHO)-K1 cells (CHO/horPDPN), and hybridomas producing mAbs against horPDPN were screened using flow cytometry. One of the mAbs, PMab-219 (IgG_2a, kappa), specifically detected CHO/horPDPN cells via flow cytometry and recognized horPDPN protein using Western blotting. Furthermore, PMab-219 strongly stained CHO/horPDPN via immunohistochemistry. These findings suggest that PMab-219 is useful for investigating the function of horPDPN.     

Synthesis and evaluation of a [18F]formyl–Met–Leu–Phe derivative: A positron emission tomography imaging probe for bacterial infections

The tripeptide formyl–Met–Leu–Phe (fMLF) is a prototype of N-formylated chemotactic peptides for neutrophils owing to its ability to bind and activate the G protein-coupled formyl peptide receptor (FPR). Here, we developed an ¹⁸F-labeled fMLF derivative targeting FPR as a positron emission tomography (PET) imaging probe for bacterial infections. The study demonstrates that the fMLF derivative fMLFXYk(FB)k (X?=?Nle) has a high affinity for FPR (Ki?=?0.62?±?0.13?nM). The radiochemical yield and purity of [¹⁸F]fMLFXYk(FB)k were 16% and >96%, respectively. The in vivo biodistribution study showed that [¹⁸F]fMLFXYk(FB)k uptake was higher in the bacterial infected region than in the non-infected region. We observed considerably higher infection-to-muscle ratio of 4.6 at 60?min after [¹⁸F]fMLFXYk(FB)k injection. Furthermore, small-animal PET imaging studies suggested that [¹⁸F]fMLFXYk(FB)k uptake in the bacterial infected region was clearly visualized 60?min after injection.     

Growth factor-dependent branching of the ureteric bud is modulated by selective 6-O sulfation of heparan sulfate

Heparan sulfate proteoglycans (HSPGs) are found in the basement membrane and at the cell-surface where they modulate the binding and activity of a variety of growth factors and other molecules. Most of the functions of HSPGs are mediated by the variable sulfated glycosaminoglycan (GAG) chains attached to a core protein. Sulfation of the GAG chain is key as evidenced by the renal agenesis phenotype in mice deficient in the HS biosynthetic enzyme, heparan sulfate 2-O sulfotransferase (Hs2st; an enzyme which catalyzes the 2-O-sulfation of uronic acids in heparan sulfate). We have recently demonstrated that this phenotype is likely due to a defect in induction of the metanephric mesenchyme (MM), which along with the ureteric bud (UB), is responsible for the mutually inductive interactions in the developing kidney (Shah et al., 2010). Here, we sought to elucidate the role of variable HS sulfation in UB branching morphogenesis, particularly the role of 6-O sulfation. Endogenous HS was localized along the length of the UB suggesting a role in limiting growth factors and other molecules to specific regions of the UB. Treatment of cultures of whole embryonic kidney with variably desulfated heparin compounds indicated a requirement of 6O-sulfation in the growth and branching of the UB. In support of this notion, branching morphogenesis of the isolated UB was found to be more sensitive to the HS 6-O sulfation modification when compared to the 2-O sulfation modification. In addition, a variety of known UB branching morphogens (i.e., pleiotrophin, heregulin, FGF1 and GDNF) were found to have a higher affinity for 6-O sulfated heparin providing additional support for the notion that this HS modification is important for robust UB branching morphogenesis. Taken together with earlier studies, these findings suggest a general mechanism for spatio-temporal HS regulation of growth factor activity along the branching UB and in the developing MM and support the view that specific growth factor-HSPG interactions establish morphogen gradients and function as developmental switches during the stages of epithelial organogenesis (Shah et al., 2004).     

Construction of physical map and mapping of chromosomal virulence genes of the biovar 3 Agrobacterium (Rhizobium vitis) strain K-Ag-1

Most plant pathogenic Agrobacterium strains have been classified into three biovars, "biovar 1 (A. tumefaciens; Rhizobium radiobacter), biovar 2 (A. rhizogenes; R. rhizogenes) and biovar 3 (A. vitis; R. vitis)". The bacteria possess diverse types of genomic organization depending on the biovar. Previous genomic physical maps indicated difference in location of rDNA and chromosomally-coded virulence genes between biovar 1 and 2 genomes. In order to understand biovar 3 genome and its evolution in relation to the biovar 1, 2 and 3 genomes, we constructed physical map of a pathogenic biovar 3 strain K-Ag-1 in this study. Its genome consisted of two circular chromosomes (3.6 and 1.1 Mbp in length), and three plasmids (560, 230 and 70 kbp). Gene mapping based on the physical map showed presence of two rDNA loci in the larger chromosome and at least one rDNA locus in the smaller chromosome. Six chromosomal virulence genes, namely chvA, chvD, chvE, glgP, exoC and ros were found in the larger chromosome and not in the smaller chromosome. The location of rDNA loci is similar with that of biovar 1 genome, whereas the location of chromosomal virulence genes is similar with that of biovar 2 genome despite of the closer 16S-rRNA based phylogenetic relation of biovar 3 with biovar 1 than with biovar 2. Genomic PFGE RFLP analysis revealed that the K-Ag-1 strain, which was isolated on a kiwifruit plant in Japan, has the closest intra-species relation with two strains isolated from grapevine plants in Japan among eight biovar 3 strains examined. This datum suggests that the line of the strain is a major one in biovar 3 in Japan. Evolution of the genome of the strain is discussed based on the data.     

A novel approach and protocol for discovering extremely low-abundance proteins in serum

The proteomic analysis of serum (plasma) has been a major approach to determining biomarkers essential for early disease diagnoses and drug discoveries. The determination of these biomarkers, however, is analytically challenging since the dynamic concentration range of serum proteins/peptides is extremely wide (more than 10 orders of magnitude). Thus, the reduction in sample complexity prior to proteomic analyses is essential, particularly in analyzing low-abundance protein biomarkers. Here, we demonstrate a novel approach to the proteomic analyses of human serum that uses an originally developed serum protein separation device and a sequentially linked 3-D-LC-MS/MS system. Our hollow-fiber-membrane-based serum pretreatment device can efficiently deplete high-molecular weight proteins and concentrate low-molecular weight proteins/peptides automatically within 1 h. Four independent analyses of healthy human sera pretreated using this unique device, followed by the 3-D-LC-MS/MS successfully produced 12 000-13 000 MS/MS spectra and hit around 1800 proteins (>95% reliability) and 2300 proteins (>80% reliability). We believe that the unique serum pretreatment device and proteomic analysis protocol reported here could be a powerful tool for searching physiological biomarkers by its high throughput (3.7 days per one sample analysis) and high performance of finding low abundant proteins from serum or plasma samples.     

Flavonoid composition of fruit tissues of citrus species

An HPLC analysis was performed on the concentrations of flavonoids in 42 species and cultivars of the Citrus genus and those of two Fortunella and one Poncirus species according to the classification system established by Tanaka. The composition of 8 flavanones and 9 flavone/ols for these species was determined in the albedo, flavedo, segment epidermis and juice vesicle tissues, and those in the fruit and peel tissues were calculated from the composition data of the tissues. A principal component analysis showed that such neohesperidosyl flavonoids as neoeriocitrin, naringin, neohesperidin, and rhoifolin had large factor loading values in the first principal component for each tissue. The flavonoid composition of citrus fruits was approximately the same within each section of Tanaka's system, except for the species in the Aurantium section and those with a peculiar flavonoid composition such as Bergamot (C. bergamia), Marsh grapefruit (C. paradisi), Sour orange (C. aurantium), and Shunkokan (C. shunkokan). The Aurantium section included both naringin-rich and hesperidin-rich species.     

Mechanisms to avoid photoinhibition in a desiccation-tolerant cyanobacterium, Nostoc commune

A desiccation-tolerant cyanobacterium, Nostoc commune, showsunique responses to dehydration. These responses are: (i) lossof PSII activity in parallel with the loss of photosynthesis;(ii) loss of PSI activity; and (iii) dissipation of light energyabsorbed by pigment–protein complexes. In this study,the deactivation of PSII is shown to be important in avoidingphotoinhibition when the Calvin–Benson cycle is repressedby dehydration. Furthermore, our evidence suggests that dissipationof light energy absorbed by PSII blocks photoinhibition understrong light in dehydrated states.     

Cytomorphologic and immunocytochemical characteristics of reactive renal tubular cells in renal glomerular disease

OBJECTIVE: To investigate the morphologic characteristics and immunocytochemical reaction to vimentin of the reactive renal tubular cells (RRTCs) in renal glomerular disease. STUDY DESIGN: We prospectively evaluated the urine cytology of renal glomerular disease in 40 patients who underwent renal biopsy. The cytology and renal biopsy specimens were analyzed for vimentin immunostaining. RESULTS: A total of 40 urine samples from the 40 patients were cytologically analyzed, and RRTCs were found in 25 samples (25 of 40, 62.5%). These RRTCs showed clear or vacuolated cytoplasm, intracytoplasmic pigmented granules (hemosiderin or lipofuscin) and large nuclei with round to irregular nuclear contours and prominent nucleoli. These cells were seen singly and in acinar clusters. Occasionally RRTCs were embedded in a cast (RRTC cast). Immunocytochemicalstudy revealed RRTCs to be positive for vimentin (25 of 25, 100%). CONCLUSION: Frequently observed characteristic cytomorphologic features of RRTCs included RRTC cast, acinar cluster, vacuolated cytoplasm and intracytoplasmic pigmented granules. A diagnosis of RRTCs can be suggested based on these cytomorphologic features. However, a definitive diagnosis will require immunocytochemical confirmation for vimentin.     

Thalassiocyclus pankensis sp. nov., a new diatom from the Panke Swamp, northern Japan (Bacillariophyta)

Thalassiocyclus pankensis sp. nov. is described from Panke Swamp (Panke-numa) located in Horonobe Town, Hokkaido, northern Japan. The species is characterized by a strong transversely undulate central area of the valve face with one to three fultoportulae on the elevated part and one fultoportula on the depressed part. The external valve face has costate radial interfascicles and very wide fascicles on the marginal area. Thickened openings of mantle fultoportulae are located at the end of interfascicles with roofed board spines. The cingulum consists of five bands. The new species is compared to the only other species belonging to genus Thalassiocyclus , T. lucens (Hustedt) Håkansson & Mahood. Thalassiocyclus pankensis is the second species in the genus Thalassiocyclus and also the first report of a Thalassiocyclus species in Japan.