Development of Micropipette Method for Collecting Vacuolar Contents from Intact Tannin Cells in Persimmon (Diospyros kaki) Fruit and Preliminary Analysis of its Constituents

In order to study the nature of tannins in vivo, we developed a method for collecting the vacuolar contents from intact tannin cells in persimmon fruit. We used a micropipette controlled with a MMS-77 micromanipulator system (Shimadzu Co., Kyoto, Japan) under an inverted microscope. Fruit flesh of mature persimmon fruit (cv. Miyazakimukaku) was cut into 300-μm-thick sections with a DSK-100 miaoslicer (Dosaka EM, Kyoto, Japan). The sections were then put on a glass slide, and a micropipette was inserted into a tannin cell to withdraw its contents. After determination of the sap volume collected, the sample was injected into a 25-μl drop of water on a glass slide. Then, the water-drop containing the tannin sample was transferred to a small microfuge tube and stored in a freezer until analysis. Based on calculations, we could collect approximately 7 to 12 nl of vacuolar contents per tannin cell. When tannin and sugar contents per tannin cell were determined, we found that tannin cells contain tannins at 10% to 15% as catechin equivalents (w/v) and 8% to 10% total sugars (w/v), while a whole fruit contains tannins at 1% to 1.5% as catechin equivalents and 10% to 13% total sugars on fresh weight basis. We are currently continuing more detailed analysis of tannin cell constituents.

Acetonitrile is better than ethanol as a dehydrating agent for cells prepared for SEM

Acetonitrile has been shown to be a better dehydrating agent than ethanol for whole tissue prepared for TEM studies. In this paper we show that it is also a better choice for dehydration of cell cultures prepared for SEM studies. The procedures used for the isolation and culture of cardiac cells in the present study have been outlined in detail by Schroedl and Hartzell (1983) and Freerksen, et al. (1984). Cells were cultured in suspension using HARV-Bioreactors (Synthecon, Inc., Friendswood, TX) for 24 hours to allow attachment and then fed each subsequent 48 hours. After a total of six days in suspension culture, cell/bead clusters were collected and washed with PBS.Primary fixation was done at 4°C, all other steps were performed at room temperature. The buffer used in all cases was 0.1M cacodylate, pH 7.3. The cell/bead cultures were processed according to the following protocol. All of the beads were placed in a 1.5 ml microfuge tube and fixed in 2% buffered glutaraldehyde overnight followed by two 5 min washes in buffer, postfixation in 1% buffered OsO4 for 90 min and three washes in buffer. One half of the beads were then placed in another microfuge tube and dehydrated in acetonitrile, while the remaining beads were dehydrated in ethanol.

Reproductive organ blood flow measured using radioactive microspheres in diestrous and estrous mice

Blood flow is a primary mechanism controlling reproductive organ functions. In the present study, radioactive microsphere techniques were adapted to measure ovarian, uterine, and vaginal blood flow levels in C57BL mice. Anesthetized animals were tracheostomized and the left carotid artery was cannulated. The heart was exposed and 113Sn-labeled spheres (15 microns size, 2 microCi, 0.1 ml) were injected via the left ventricle. Reference sample was obtained by carotid artery blood "free flowing" into a tared microfuge tube for 1 min. The animal was killed, and selected tissues were excised for weighing and radioactivity measurement to determine flow. Absence of differences in flow levels (ml.min-1.g-1) to paired nonreproductive organs (adrenals and kidneys) validated the procedure. Blood flow levels were significantly higher in the ovaries, but not in the uterus and vagina of estrous mice vs. diestrous mice. Comparison of left vs. right ovaries suggested consistent blood flow distribution during diestrus. Ovarian blood flow level is enhanced during estrus and, in addition, is highly nonuniform regarding right-left flow distribution. Nonuniform ovarian blood flow distribution in estrous mice leads us to speculate that alternating right-left (i.e. nonuniform) ovulation predominates during each murine estrous cycle.

Counting of Beta and Gamma Emitters Used in Clinical Radioimmunoassays and Competitive-Binding Assays by Liquid Scintillation Counters

Although liquid scintillation (LS) counters were originally designed to count low-energy beta emitting isotopes, they can also be used to count gamma emitters used in radioimmunoassay. This is made possible by placing the gamma emitter inside a "Microfuge" tube suspended from a LS vial cap into a scintillator fluid containing an electron density increaser. Efficiencies of 71% are obtained for 125I by this method, comparable to that obtained with Nal (TI) crystal gamma counters. Efficiencies for other clinically important isotopes used in radioimmunoassay—131I, 57Co, 60Co, 51Cr, 59Fe—by the Microfuge method are 15-19%; therefore, for these isotopes a LS "Mini-Vial" method is suggested, in which smaller volumes may be used. The dual capability of LS counters to count both beta and gamma emitters extends its usefulness in the clinical laboratory.