Hexokinase Levels in Discrete Regions of Rat Brain: Evaluation by Quantitative Immuno fluoresence Using Interactive Laser Cytometry and Comparison with Basal Rates of Glucose Utilization

Relative levels of hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) have been determined in 16 discrete regions of adult rat brain by a quantitative immunofluorescence method. The distribution of immunofluorescence in brain sections was determined by interactive laser cytometry and related to hexokinase content by comparison with standard sections containing known amounts of the enzyme. In many of these regions, referred to here as group I regions, hexokinase content was correlated with previously reported basal rates of glucose utilization. However, several regions (group II regions) in which hexokinase content exceeded that expected from basal rates of glucose utilization were also detected. Compared with the corresponding regions from albino rat brain, higher hexokinase levels were found in the dorsal and ventral lateral geniculate (group I regions) of pigmented Norway rats, a result reflecting previously reported increased glucose utilization by these regions in pigmented rats. There was no difference in hexokinase levels in the superior colliculus, a group II structure, from albino and pigmented rats, a finding implying that a reported increase in rate of glucose utilization in the superior colliculus of pigmented rats is effected without an increase in hexokinase content. It is suggested that group II regions may be adapted to sustain increases in rates of glucose utilization that are, relative to basal rates, considerably greater than those experienced by group I regions.     

Evaluation of chemical methods for estimating available zinc and response of green gram (Phaseolus aureus roxb) to applied zinc in non-calcareous soils

Summary Studies were conducted in 22 non-calcareous soils (India) to evaluate various extractants,viz. (6N HCl, 0.1N HCl, EDTA (NH₄)₂CO₃, EDTA NH₄OAc, DTPA+CaCl₂ and 1M MgCl₂) to find critical levels of soil and plant Zn for green gram (Phaseolus aureus Roxb.). The order of extractability by the different extractants was 6N HCl>0.1N HCl>EDTA (NH₄)₂CO₃<EDTA NH₄OAc DTPA+CaCl₂>1M MgCl₂. Critical levels of 0.48 ppm DTPA × CaCl₂ extractable Zn, 0.80 ppm EDTA NH₄OAc extractable Zn, 0.70 ppm EDTA (NH₄)₂CO₃ extractable Zn, and 2.2 ppm 0.1N HCl extractable Zn were estimated for the soils tested. The critical Zn concentration in 6 weeks old plants was found to be 19 ppm. The 0.1N HCl method gave the best correlation (r=0.588^**) between extractable Zn and Bray's per cent yield, while with DTPA+CaCl₂, it was slightly low (r=0.542^**). The DTPA + CaCl₂ method gave significant (r=0.73^**) correlation with plant Zn concentration. The 0.1N HCl gave the higher correlation with Zn uptake (r=0.661^**) than DTPA (r=0.634^**) 6N HCl and 1M MgCl₂ method gave nonsignificant positive relationship with Bray's per cent yield. For noncalcareous soils apart from the common use of DTPA+CaCl₂, 0.1N HCl can also be used for predicting soil available Zn. The use of 0.1N HCl would be much cheaper than DTPA and other extractants used in the study.     

Inhibition of selected fungi by bacterial isolates fromTripsacum dactyloides L.

Summary In an incubation study addition of green manure caused a reduction in the ammonia volatilized initially from both sodic and reclaimed soils but extensive volatilization occurred from the sodic soil, amended with green manure, after the tenth day till the conclusion of the experiment after 9 weeks. Volatilization from the reclaimed soil was much less. There was a slight build up of organic carbon and ammoniacal nitrogen in both the soils though greater in the reclaimed soil. More of nitrate and nitrite accumulated in the sodic soil.     

Laser microirradiation of kinetochores in mitotic PtK2 cells

Irradiation of the kinetochore region of PtK₂ chromosomes by laser light of 532 nm was used to study the function of the kinetochore region in chromosome movement and to create artificial micronuclei in cells. When the sister kinetochores of a chromosome were irradiated at prometaphase, the affected chromosome detached from the spindle and exhibited no further directed movements for the duration of mitosis. The chromatids of the chromosome remained attached to one another until anaphase, at which point they separated. No poleward movement of the chromatids was observed, and at telophase they passively moved to one of the daughter cells and were enclosed in a micronucleus. The daughter cell containing the micronucleus was then isolated by micromanipulation and followed through subsequent mitoses. At the next mitosis, two chromosomes, each with two chromatids, condensed in the micronucleus. These chromosomes did not attach to the spindle and showed chromatid separation, but no poleward movements at anaphase. They were again enclosed in micronuclei at telophase. The third generation mitosis was similar to the second. Occasionally, both the irradiation-produced and naturally occurring micronuclei exhibited no chromosome condensation at mitosis. Feulgenstained monolayers of PtK₂ cells with naturally occurring micronuclei showed that some micronuclei stain positive for DNA and others do not. This finding raises questions about the fate of chromosomes in a micronucleus.     

The measurement of subnanosecond fluorescence decay of flavins using time-correlated photon counting and a mode-locked Ar ion laser.

A system is described consisting of a mode-locked Ar ion laser and time-resolved photon-counting electronics. The system is capable of measuring fluorescence lifetimes in the subnanosecond time domain. The Ar ion laser is suitable for the excitation of flavins, since the available laser wavelengths encompass the first absorption band of the yellow chromophore. Due to the high radiation density and the short pulse, both the time and wavelength resolution of the fluorescence of very weakly emitting compounds can be measured. Experiments have been described for flavin models exhibiting single and multiple modes of decay. In these examples lifetimes were determined both from deconvolved decay curves and from direct analysis of the tail of the curve, where no interference of the exciting pulse is encountered. Both determinations showed very good agreement. Due to the highly polarized laser light the decay of the emission anisotropy could be measured directly after the exciting pulse. In principle, fast rotational motions might be detected. An anisotropy measurement conducted with a flavoprotein with a noncovalently attached FAD is presented.     

A New Approach Using the SYBR Green-Based Real-Time PCR Method for Detection of Soft Rot Pectobacterium odoriferum Associated with Kimchi Cabbage

Pectobacterium odoriferum is the primary causative agent in Kimchi cabbage soft-rot diseases. The pathogenic bacteria Pectobacterium genera are responsible for significant yield losses in crops. However, P. odoriferum shares a vast range of hosts with P. carotovorum, P. versatile, and P. brasiliense, and has similar biochemical, phenotypic, and genetic characteristics to these species. Therefore, it is essential to develop a P. odoriferum-specific diagnostic method for soft-rot disease because of the complicated diagnostic process and management as described above. Therefore, in this study, to select P. odoriferum-specific genes, species-specific genes were selected using the data of the P. odoriferum JK2.1 whole genome and similar bacterial species registered with NCBI. Thereafter, the specificity of the selected gene was tested through blast analysis. We identified novel species-specific genes to detect and quantify targeted P. odoriferum and designed specific primer sets targeting HAD family hydrolases. It was confirmed that the selected primer set formed a specific amplicon of 360 bp only in the DNA of P. odoriferum using 29 Pectobacterium species and related species. Furthermore, the population density of P. odoriferum can be estimated without genomic DNA extraction through SYBR Green-based real-time quantitative PCR using a primer set in plants. As a result, the newly developed diagnostic method enables rapid and accurate diagnosis and continuous monitoring of soft-rot disease in Kimchi cabbage without additional procedures from the plant tissue.     

Preliminary screening of antioxidant and cytotoxic potential of green seaweed,Halimeda opuntia (Linnaeus) Lamouroux

Marine natural products have displayed numerous advantageous effects on biological activities, including antioxidants and cytotoxicity. The total lipids, carotenoids, chlorophyll a and b content, total phenolic content (TPC), total flavonoid content (TFC), and antioxidant activity of methanolic crude extract of the green seaweed Halimeda opuntia were all measured in this study. The TPC of the extracts was determined according to the Folin-Ciocalteu method, yielding a result of 55.04 ± 0.98 mg GAE/g of extract. As determined by the aluminium chloride colorimetric method, the TFC of the extract was 40.02 ± 0.02 mg QE/g of extract. Antioxidant activity was determined by using a 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay with different concentrations that ranged between 200 and 1000 µg/mL, noted H. opuntia as the highest in DPPH reduction (63.61 %) at 1000 µg/mL concentration. Total antioxidant capacity (TAC) of the extract was 57.36 ± 0.004 mg AAE/g extract at concentration of 1.0 mg/mL. The cytotoxic activity of this seaweed was pre-screened against a panel of cell lines including estrogen receptor-positive human breast adenocarcinoma (MCF-7), estrogen negative human breast adenocarcinoma (MDA-MB-231), human colorectal adenocarcinoma (HT-29), human hepatocellular carcinoma (HepG2), and mouse embryonic fibroblast (3T3) using the MTT assay. The content of total lipids in H. opuntia was 1.60 ± 0.002 %. Total carotenoids were 115.57 ± 0.98 µg/g, while chlorophyll a and b were 148.73 ± 2.60 µg/g and 290.83 ± 9.46 µg/g, respectively. In terms of cytotoxicity activity, methanolic extract of H. opuntia was found to be highly cytotoxic to MCF-7 cells, with an IC₅₀ of 25.14 ± 1.02 g/mL, and slightly less so to 3T3 cells (IC₅₀ 65.23 ± 0.25 µg/mL). This study's findings suggest that natural pigments (carotenoids and chlorophyll), phytochemicals like phenolic and flavonoid compounds found in this species may play an important role and could be used as a natural cancer treatment.     

Green fluorescent protein-tagged Edwardsiella tarda reveals portal of entry in fish

The application of green fluorescent protein (GFP) to identify the portal(s) of entry of bacterial pathogens in animal hosts was studied using the fish pathogen Edwardsiella tarda and blue gourami, Trichogaster trichopterus. An immersion challenge model was utilized to mimic natural infection conditions in fish. Gastrointestinal tract, gills and the body surface of fish were found to be the sites of entry of virulent E. tarda (PPD130/91) by histological and infection kinetics studies. On the other hand, avirulent E. tarda (PPD125/87) was mainly found in the gastrointestinal tract, and the bacterial population in tissue declined over a period of 7 days.     

Confocal fluorescence microscopy of plant cells

Summary The confocal laser scanning microscope (CLSM) has become a vital instrument for the examination of subcellular structure, especially in fluorescently stained cells. Because of its ability to markedly reduce out-of-focus flare, when compared to the conventional wide-field fluorescence microscope, the CLSM provides a substantial improvement in resolution along the z axis and permits optical sectioning of cells. These developments have been particularly helpful for the investigation of plant cells and tissues, which because of their shape, size, and optical properties have been difficult to analyze at high resolution by conventional means. We review the contribution that the CLSM has made to the study of plant cells. We first consider the principle of operation of the CLSM, including a discussion of image processing, and of lasers and appropriate fluorescent dyes. We then summarize several studies of both fixed and live plant cells in which the instrument has provided new or much clearer information about cellular substructure than has been possible heretofore. Attention is given to the visualization of different components, including especially the cytoskeleton, endomembranes, nuclear components, and relevant ions, and their changes in relationship to physiological and developmental processes. We conclude with an effort to anticipate advances in technology that will improve and extend the performance of the CLSM. In addition to the usual bibliography, we provide internet addresses for information about the CLSM.