Label-free calcium imaging in ischemic retinal tissue by TOF-SIMS

The distribution and movement of elemental ions in biologic tissues is critical for many cellular processes. In contrast to chemical techniques for imaging the intracellular distribution of ions, however, techniques for imaging the distribution of ions across tissues are not well developed. We used time-of-flight secondary ion mass spectrometry (TOF-SIMS) to obtain nonlabeled high-resolution analytic images of ion distribution in ischemic retinal tissues. Marked changes in Ca²⁺ distribution, compared with other fundamental ions, such as Na⁺, K⁺, and Mg²⁺, were detected during the progression of ischemia. Furthermore, the Ca²⁺ redistribution pattern correlated closely with TUNEL-positive (positive for terminal deoxynucleotidyl transferase-mediated 2′-deoxyuridine 5′-triphosphate nick end-labeling) cell death in ischemic retinas. After treatment with a calcium chelator, Ca²⁺ ion redistribution was delayed, resulting in a decrease in TUNEL-positive cells. These results indicate that ischemia-induced Ca²⁺ redistribution within retinal tissues is associated with the order of apoptotic cell death, which possibly explains the different susceptibility of various types of retinal cells to ischemia. Thus, the TOF-SIMS technique provides a tool for the study of intercellular communication by Ca²⁺ ion movement.     

Cytochemical investigation of genic male-sterility in Chinese cabbage

A genic male sterile Chinese cabbage, Brassica campestris L. ssp. chinensis Makino, was examined using cytological and cytochemical methods to characterize the process of pollen abortion in this plant. Thick sections of both fertile and sterile anthers at different developmental stages were stained using Toluidine Blue O, Periodic Acid-Schiff’s (PAS) reaction and Sudan Black B to detect cytochemical changes that may occur in the distribution of insoluble polysaccharide and lipid storage bodies. Pollen abortion in sterile anthers occurs at an early stage of microspore development. During early microspore development, reductions in the number of starch grains in the connective tissue of fertile anthers coincide with the accumulation of starch grains in cells of the anther wall. In the late microspore stage, a large vacuole forms in the microspore, and tapetal cells synthesize and accumulate lipid droplets. The cellular organization of tapetal cells in sterile anthers appears similar to that in fertile anthers, except for the absence of lipid droplets in cells of sterile anthers and diffusely labeled tapetal polysaccharides, suggesting defects in nutrient storage. Supported by National Natural Science Foundation of CHINA (30170060)     

Cultivar variability in the Agrobacterium-rice cell interaction and plant regeneration

Thirteen cultivars of rice ( Oryza sativa ) were tested for plant regeneration from calli initiated from the scutella of mature seeds by water stress treatment using a high concentration of agarose, and examined for their response to Agrobacterium tumefaciens LBA4404, carrying a plasmid pTOK233, harboring genes for kanamycin resistance ( npt II), hygromycin resistance ( hpt ) and β -glucuronidase ( gus ). Plant regeneration frequency was considerably increased in most of the cultivars when the calli were treated with water stress, as compared with untreated controls. In particular, the cultivars Dongjinbyeo, IR43, Nagdongbyeo and Sinseonchalbyeo showed an increased frequency of shoot regeneration. Expression of GUS was detected in all of the co-cultivated cultivars. Based on GUS expression at 3 days after co-cultivation with A. tumefaciens , three rice cultivars (Dongjinbyeo, Hwayoungbyeo and Nagdongbyeo) were judged highly susceptible to A. tumefaciens , while Milyang 23, Nonganbyeo and Samgangbyeo cultivars were weakly susceptible. Plantlets were readily regenerated when the hygromycin-resistant calli were transferred to a regeneration medium containing hygromycin. Intense blue staining was observed in GUS assays of leaf segments, roots and flower organs from regenerated plants. Stable integration and expression of the introduced hpt and gus genes were confirmed by Southern blot analysis of the transformants. Therefore, Dongjinbyeo and Nagdongbyeo cultivars proved to be both highly susceptible to A. tumefaciens and highly responsive to plant regeneration.     

Monoclonal antibodies for intravesical radioimmunotherapy of human bladder cancer

Local administration of radioimmunoconjugates may allow successful tumor therapy. Bladder cancer appears well suited to this approach, because of its superficial and multifocal nature, and because it will allow direct intravesical administration of conjugates. Implantation of human bladder cancer cell lines in the bladder wall of nude rats results in tumor formation, providing an excellent model to test this. We have developed two murine monoclonal antibodies (MAbs), BLCA-8, IgG₃, and BLCA-38, IgG₁, both of which react with malignant cells and shed into voided urine of patients with transitional cell carcinoma (TCC) of the bladder, but not with normal bladder urothelial cells. Radioimmunoconjugates produced with¹³¹Iodine (¹³¹I) or¹²⁵I have been used for biodistribution studies following administration directly into the bladder. Radioiodinated intact MAbs or Fabs administered intravesically into nontumor-bearing rats did not leak into the systemic circulation and were stable in urine for up to 100h. Biodistribution studies carried out following intraperitoneal or intravesical administration of radioimmunoconjugates to tumor-bearing nude rats indicate good tumor uptake of both MAbs. Together with immunoreactivity assays, these studies demonstrate that¹³¹I-labeled MAbs have considerable potential for intravesical radioimmunotherapy of human bladder tumors, and further studies are under way.     

The retinoblastoma susceptibility gene product undergoes cell cycle-dependent dephosphorylation and binding to and release from SV40 large T

Synchronized monkey cells pulse-labeled with [35S]-methionine and chased for various lengths of time were extracted, and immunoprecipitations were performed using monoclonal antibodies directed against the retinoblastoma protein (RB) and SV40 T antigen (T). By following a discrete population of these two proteins through the cell cycle, the following information was obtained. RB, which is wholly unphosphorylated in G1, became phosphorylated at the beginning of S and remained phosphorylated through S and G2. RB was, then, completely dephosphorylated between the end of G2 and the beginning of G1. Second, while all of the detectable unphosphorylated RB can be found complexed with T, these complexes present during G1 dissociated in S and reformed again in M or early G1. Finally, T molecules appeared to oligomerize prior to binding RB. Thus, complex formation between T and RB may be regulated in part by the cell cycle-dependent phosphorylation and dephosphorylation of RB and by the quaternary structure of T.     

Extracellular signal-regulated kinase 1/2 plays a pro-life role in experimental brain stem death via MAPK signal-interacting kinase at rostral ventrolateral medulla

Background  

As the origin of a life-and-death signal detected from systemic arterial pressure, which sequentially increases (pro-life) and decreases (pro-death) to reflect progressive dysfunction of central cardiovascular regulation during the advancement towards brain stem death in critically ill patients, the rostral ventrolateral medulla (RVLM) is a suitable neural substrate for mechanistic delineation of this fatal phenomenon. The present study assessed the hypothesis that extracellular signal-regulated kinase 1/2 (ERK1/2), a member of the mitogen-activated protein kinases (MAPKs) that is important for cell survival and is activated specifically by MAPK kinase 1/2 (MEK1/2), plays a pro-life role in RVLM during brain stem death. We further delineated the participation of MAPK signal-interacting kinase (MNK), a novel substrate of ERK in this process.     

Disturbance-mediated competition: the interacting roles of inundation regime and mechanical and herbicidal control in determining native and invasive plant abundance

Disturbance is a key component of many successful plant invasions. However, interactions among natural and anthropogenic disturbances and effects of these interacting disturbances on invasive plants and desired vegetation are rarely examined. We investigated the effect of anthropogenic disturbance (herbicidal and mechanical) along a natural inundation gradient (20–282 days) on the biomass and resource allocation of the invasive wetland plant, alligator weed (Alternanthera philoxeroides), and two co-occurring competitor plants, the introduced grass, kikuyu (Pennisetum clandestinum), and the native grass, couch (Cynodon dactylon), over a 2-year period. In the absence of additional disturbance, kikuyu biomass was negatively affected, alligator weed biomass was positively affected, and couch biomass was not affected by inundation disturbance. In addition, kikuyu was not affected by the selective removal of alligator weed, while couch increased in wetter habitats where kikuyu was absent due to inundation stress. This suggests that kikuyu is a superior competitor in drier habitats and inundation facilitates the invasion of alligator weed, while couch is an inferior competitor to both kikuyu and alligator weed and is therefore suppressed across its entire niche by these two introduced species. Mowing alone had no effect on the biomass of the species, suggesting the plants are equally tolerant of shoot removal. Selective herbicide reduced alligator weed biomass by 97.5% and the combination of selective herbicide and mowing reduced the biomass of alligator weed significantly more than herbicide alone, by 98.6% compared with un-manipulated controls. To predict community change and prevent sequential exotic plant invasions after weed removal, it is necessary to consider the interacting effects of disturbance and the niche space of invasive species in the local propagule pool.     

Production of biohydrogen by recombinant expression of [NiFe]-hydrogenase 1 in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background  

Hydrogenases catalyze reversible reaction between hydrogen (H₂) and proton. Inactivation of hydrogenase by exposure to oxygen is a critical limitation in biohydrogen production since strict anaerobic conditions are required. While [FeFe]-hydrogenases are irreversibly inactivated by oxygen, it was known that [NiFe]-hydrogenases are generally more tolerant to oxygen. The physiological function of [NiFe]-hydrogenase 1 is still ambiguous. We herein investigated the H₂ production potential of [NiFe]-hydrogenase 1 of Escherichia coli in vivo and in vitro. The hya A and hya B genes corresponding to the small and large subunits of [NiFe]-hydrogenase 1 core enzyme, respectively, were expressed in BL21, an E. coli strain without H₂ producing ability.