A NEW CLEARING-SQUASH TECHNIQUE FOR THE STUDY OF OVULE DEVELOPMENT IN ANGIOSPERMS

The simple, efficient method described here for the study of ovule and megagametophyte development in angiosperms provides for the extension of investigation beyond the limits imposed by the traditional but arduous section technique. Excised pistils previously fixed in FPA₅₀ and stored in 70 % ethanol are placed in a clearing fluid composed of lactic acid (85 %), chloral hydrate, phenol, clove oil, and xylene (2:2:2:2:1, by weight). After 24 hr, ovules dissected from the ovularies are transferred with some of the fluid to a slide, covered so that the cover glass is supported laterally by two permanently affixed covers, and examined with phase contrast optics. The unique action of the clearing fluid permits the study of cellular structure with the phase oil objective focused at any focal plane within the ovule. Downward focusing thus reveals a series of optical sections in the sagittal, frontal, or transverse plane depending on the orientation of the ovule. Orientation can be altered by a slight shifting of the cover glass on the lateral support mounts. The ovules become quite fragile in the clearing fluid. Pressure applied to the cover glass gradually breaks the ovule apart without disrupting the structural integrity of individual cells. This squash procedure provides for extending observations to cytological features of megasporocytes, megaspores, and megagametophytes previously identified in intact ovules. The new method is applied here to the study of ovule development in two unrelated species, Cassia abbreviata Oliver var. granitica Bak. f. (Leguminosae) and Ludwigia uruguayensis (Camb.) Hara. (Onagraceae). For best results, the ovules of Ludwigia must be pretreated in lactic acid (85 %) for 24 hr prior to application of the clearing fluid. Other methods for pretreatment likely will be required as the technique is applied to a wider range of flowering plant species.     

A freeze-fracture study of exocytosis and reflexive gap junctions in human ovarian decidual cells

Fine-structural features of ovarian decidual cells and their mode of secretion were examined by means of freeze-fracture microscopy. Unique cortical peduncular processes contained secretory vesicles within the expanded peduncle tip, the membrane-leaflets of which exhibited a particle-poor E face adjacent to the vesicle lumen and a P face containing a greater particle number. Exocytosis from attached peduncles involved release of vesicular profiles 40-55 nm in diameter; small particles 8.5-11.5 nm in diameter were also observed at degranulation sites. In fractures revealing the E face of the plasmalemma, cytoplasmic portals at the bases of peduncular stalks were distinguishable from endocytic vesicles. The frequent occurrence of reflexive gap junctions associated with peduncles was shown by freeze-fracture. However, there appeared to be no consistent spatial relationship between gap junctions, secretory peduncles, or sites of exocytosis. Freeze-fracture analysis of the topography of reflexive gap junctional profiles revealed that such gap junctions share basic similarities with intercellular gap jum particle-free aisles. The finding in the present study of reflexive gap junctions occurring between peduncles and the cell soma, as well as between peduncles, suggests that the original definitiof the same cell should be broadened to include any gap junctional specialization formed between portions of the plasma membrane of one cell.     

Characteristics of the muscle mechanoreflex during quadriceps contractions in humans

We examined muscle sympathetic nerve activity (MSNA) in thenonexercising lower limb during repetitive static quadricepscontraction paradigm at 25% maximal voluntary contraction in eightmen. Subjects performed 20-s contractions with 5-s rest periods for upto 12 contractions. Although the workload was constant, we found that MSNA amplitude rose as a function of contraction number [0.6 ln (amplitude/min)/contraction]; this suggests chemicalsensitization of the muscle reflex response. We employedsignal-averaging techniques and then integrated the data to examine theonset latency of the MSNA response as a function of the 25-scontraction-rest period. We observed an onset latency of ~4-6 s.Moreover, although the onset latency did not appear to vary as afunction of contraction number, the rate of MSNA increase tookapproximately four contractions to reach a steady-state rate of rise;this suggests contraction-induced sensitization. The onset latencyreported here is similar to findings in recent animal studies, but itis at odds with latencies determined in prior human handgripcontraction studies. We believe our data suggest that1) mechanically sensitive afferentscontribute importantly to the MSNA response to the paradigm employedand 2) these afferents may besensitized by the chemical products of muscle contraction.     

Type-A retrovirus particles in heterohybridomas: precaution for human monoclonal antibody purification

Fine structural features of the murine myeloma MHFP-1 and two heterohybridomas secreting human IgM monoclonal antibody were examined. Intracisternal type-A retrovirus particles were found in both MHFP-1 and the heterohydribomas constructed by fusing MHFP-1 and human peripheral blood lymphocytes. The implications of this finding for the purification of human monoclonal antibody for therapeutic applications is discussed.     

Coronary artery embolization in closed-chest canines using flexible radiopaque plugs

A new technique induces localized myocardial infarction in closed-chest dogs by placing discrete plugs in coronary arteries without using cumbersome coaxial catheters or guide wires. Flexible plugs, essential to this method, are formed by extruding a dental impression polymer, rendered radiopaque with sodium iodide, into spaghetti-like strands. Segments of these strands can be injected through a catheter into a selected coronary artery. Contact with blood or saline causes plugs to swell. The mean increase in plug diameter due to swelling was 27 +/- 20%. Eight anesthetized dogs were embolized via carotid approach [6 left anterior descending (LAD), 1 left circumflex (LCX), and 1 LAD and LCX]. Plug positions were monitored fluoroscopically. One animal died at 2 days postembolization. The remaining seven dogs were killed after 14-37 days. Autopsies showed complete vessel occlusion and localized infarction. Infarcts resulting from coronary artery occlusion with one, two, or three plugs involved 2-26% of the left ventricular mass.     

Ten years of vegetation dynamics in two rivulets in Lower Saxony (FRG)

The vegetation dynamics in six permanent plots in two lowland rivulets of the Federal Republic of Germany are analyzed. The year-to-year change in species cover is displayed by means of tables. In each site there are core species (both hydrophytes and helophytes) which have been able to successfully reproduce within the sampling plots over the total observation period. There are also transient hydrophytes which regularly become washed in from the upper course, and transient helophytes growing permanently into the river from the banks.A numerical analysis of the performance of the 12 most frequent and abundant hydrophytes in relation to various independent variables was carried out using canonical correspondence analysis. There is no directional temporal variation within the vegetation data set. The hydrochemical variables were almost constant within the observation period. Rainfall in summer has some influence via discharge and turbidity. Most of the variance in the data set is explained by the position of the sites along the rivers. Most of the residual variance can be explained by a binary disturbance variable. The processes observed can mostly be explained from life history characteristics of the dominant species, particularySparganium emersum, Ranunculus peltatus andPotamogeton natans. The spatial scale of the study site was relatively adequate. A smaller size would have produced noisy data (suggesting erratic change), while a greater size would have produced no change at all. The adequate temporal scale for observation is the comparison of the yearly maxima because of the seasonality of most of the species. An exact prediction of dominance and species composition of the following year is impossible.     

A Bodian method for mounted frozen sections.

For application of the Bodian method to frozen sections, cut frozen peripheral nerve or muscle at 10 mum and mount. Fix for 4 days in 18 parts 80% ethanol, 1 part 10% formalin, and 1 part glacial acetic acid. Fix central nervous tissue in the same mixture prior to freezing and sectioning, and after mounting postfix for 4 days. Impregnate by the Bodian procedure. The results equal Bodian stains of paraffin sections. The technique is simple and reliable. The use of 10 mum frozen sections produces little artifact and allows alternate serial sections to be stained with other techniques.